Demonstrations of hematologic cell function in zebrafish embryos. (A-D) Using spi1:EGFP transgenic animals,58 in vivo time-lapse confocal microscopy over 30 seconds shows: (A) migration of a leukocyte (arrowhead) from the arteriole (a) and its movement between cells of the extravascular compartment toward a venule (v) (B-D). Video S1 is the movie from which these 4 still images were selected; it displays more clearly the leukocyte assuming a “dumbbell” morphology as it passes between extravascular supporting cells. (E,F) Neutrophil chemotaxis mpx-expressing neutrophils (by whole mount in situ hybridization, blue) are not abundant at the site of injury 1 hour after tail transection (E) but accumulate at the injured site after 8 hours (F, arrowhead). (G,H) By electron microscopy of 7 dpf embryos, neutrophils identified by their pathognomonic electron-dense granules are demonstrated within the vasculature (G) and muscle (H) in the vicinity of a sterile wound. (I-L) Macrophage phagocytic function. After injection of India ink, there is nonembolic accumulation of carbon particles in the ventral tail (I), within a marginated phagocytic cell (open arrowhead, J), in a field including a marginated bilobed granulocyte (K) for comparison. Dashed line divides different focal planes of the same tissue section. Electron microscopy demonstrates phagosomes (open arrowheads) within an adult splenic macrophage (L). “e” indicates an adjacent erythrocyte; I is a brightfield unstained image; J and K are hematoxylin and eosin-stained sections. (M,N) Expansion of hematopoiesis by jak2a overexpression. Normal fluorescence of the ICM (arrowhead) in Tg(spi1:EGFP) embryo at 24 hpf (M). Expansion of the ICM (arrowheads) is demonstrated after injection of constitutively active zebrafish tel-jak2a (N). Bars represent 2 μm (G,H,L). Microscopy was performed using a Bio-Rad MRC1024 confocal microscope (Bio-Rad, Hercules, CA; A-D), Siemens Elmiscope 102 transmission electron microscope (Siemens, Munich, Germany; G,H,L), Nikon SMZ-1500 microscope (Nikon) equipped with a 0.75-11.25× objective (E,F,I), a Nikon Optiphot-2 microscope equipped with a 100×/1.40 oil objective (J,K) and a Leica MZFIII fluorescence microscope (Leica, Wetzlar, Germany) equipped with a 0.8-10.0× objective (M,N). Electron microscopy images were printed to photographic paper and then digitised. Images were obtained using a Zeiss Axiocam MRc5 digital camera (Carl Zeiss) with Axiovision AC software (Release 4.5) or an Olympus DP70 digital camera (Olympus-Australia, Melbourne, Australia) with DP controller 1.2.1.108 software. Images were processed using Adobe Photoshop CS2 9.0 and Adobe Illustrator CS2 12.0.1 (Adobe Systems).
