Neutrophil differentiation is severely impaired in c-myb^hkz3/hkz3 mutants. (A-B) Loss of lyz expression in c-myb^hkz3/hkz3 mutants. WISH of lyz in 36-hpf siblings (A) and c-myb^hkz3/hkz3 mutants (B). (C-H) Myeloid progenitor, macrophage lineage, and neutrophil progenitor markers were not affected in c-myb^hkz3/hkz3 mutants as evidenced by unaltered expression of pu.1 (C-D), apoeb (E-F), and cebp1 (G-H). (I-J) mpx⁺ cells were slightly decreased in c-myb^hkz3/hkz3 mutants. WISH of mpx expression in 36-hpf siblings (I) and c-myb^hkz3/hkz3 mutants (J). (K-L) Unaltered cybb expression in c-myb^hkz3/hkz3 mutants. WISH of cybb expression in 36-hpf siblings (K) and c-myb^hkz3/hkz3 mutants (L). (M-N) Decreased srgn expression in c-myb^hkz3/hkz3 mutants. WISH of srgn expression in 36-hpf siblings (M) and c-myb^hkz3/hkz3 mutants (N). (O-P) Decreased SB⁺ cells in c-myb^hkz3/hkz3 mutants. SB staining in 36-hpf siblings (O) and c-myb^hkz3/hkz3 mutants (P). (Q-R) In vivo imaging of 2-dpf Tg(mpx:GFP) embryos by VE DIC microscopy. Left panel, Bright-field DIC image; right panel, an overlay of bright-field DIC and fluorescent image. Blue arrowheads identify granules contained by neutrophils (white arrows) expressing GFP. (S) Quantifications of WISH, SB staining, and DIC analysis for panels A to R (Student t test, **P < .01, ***P < .001, ****P < .0001; WISH and SB, n ≥ 20; DIC, n ≥ 60). (T) May-Grünwald-Giemsa staining of neutrophils in 3-dpf embryos (left) and neutrophils were quantitated by morphology (right) (Student t test, *P < .05, n ≥ 500). Scale bars, 200 μm (O-P) and 5 μm (Q, R, T).