Functional studies of granulocytes in zebrafish embryos.

(A-E) Acute inflammation assay for granulocyte function in embryonic zebrafish at 6 dpf, based on tail transection and whole-mount histochemical staining for myeloperoxidase. Whole-mount zebrafish embryos were subjected to myeloperoxidase histochemical staining before (A), immediately after (B), and 8 hours after (C) transection near the tail tip. Note the scattered population of darkly staining peroxidase-positive cells in the ventral vein region in nontraumatized embryos and the accumulation of peroxidase activity at the site of acute inflammation (B and C arrowheads). Panel D displays an array of 16 embryos, indicating the assay is highly reproducible. At higher power, under Nomarksi illumination, the aggregated myeloperoxidase activity is seen to be distinctly cellular (E). (F, G) Comparison of the pattern of myeloperoxidase histochemical staining (F) ando-dianisidine staining (G) for hemoglobin in embryos 6 hours (left tail in panels) and 2 days (right tail in panels) after tail transection, showing the longer persistence of peroxidase-positive cells compared with hemoglobin-containing cells. (H-J) Identical assay to those in panels A to C, performed in 48-hpf embryos but using whole-mount in situ hybridization for mpx-expressing cells rather than histochemistry. The pattern of mpx-expressing cells (black dots) is shown before (H), immediately after (I), and 8 hours after (J) tail transection. Note the accumulation ofmpx-expressing cells at the site of transection (shown by the black arrow) after (J), but not before (H, I), 8 hours. (K, L) Electron micrographs showing granulocytes, recognizable by their characteristic cytoplasmic cigar-shaped, electron-dense granules, in the vicinity of the trauma site in embryos subjected to tail transection 8 hours earlier. Granulocytes are seen within a vessel (K) and at an unusual site between skeletal muscle fibers (L). Scale bar = 2 μm (K, L).