Gene expression analysis

Expression data were surveyed in an online database²⁵ and confirmed by whole-mount in situ hybridization as previously described^26,27 using an antisense riboprobe corresponding to nucleotides −3 to 453 of mpeg1 cDNA sequence. Other riboprobes were csf1r²⁸ and mpx.²⁹

mpeg1 promoter identification and cloning

Zebrafish genome assembly Zv8 (http://www.ensembl.org/Danio_rerio/) informed primer design for amplification of 30437143–30438999 nt from chromosome 8, generating 1.86 kb of sequence corresponding to the immediately proximal mpeg1 5′-untranslated region. Cloning used Gateway methods and vectors.³⁰ Supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article) lists promoter and riboprobe primer sequences.

Microinjection and transgenesis

mpeg1 transgenesis was performed using Gateway protocols for Tol2-mediated recombination.³⁰ Antisense morpholino oligonucleotides were obtained from GeneTools (www.gene-tools.com) and microinjected at 200 to 500μM into 1- or 2-cell embryos at 1.7 nL per bolus (morpholino oligonucleotide sequences in supplemental Table 1).

Leukocyte function studies

For wounding, tails were transected coronally by scalpel blade at 3 dpf²⁹ and embryos mounted in 1.5% low melting agarose. Imaging commenced approximately 4 minutes after injury. For neutral red staining, embryos were incubated in egg water with 2.5 μg/mL neutral red for 12 hours.³¹ To demonstrate phagocytosis, Penicillium marneffei spores were prepared as described.³² Spores were heat-killed at 70°C for 15 minutes. Calcofluor staining was performed by incubation of spores in 10mM Calcofluor White (Sigma-Aldrich) for 15 minutes, followed by several rounds of washing and resuspension in normal saline. A total of 10 to 20 spores/embryo were microinjected into the somatic muscle of 3 dpf embryos and imaging commenced 20 minutes later.

Microscopy and image analysis

For dissecting microscopy (Figures 1, ​,2A-B,E),2A-B,E), an Olympus SZX16 microscope with a DP71 camera and 1× (Figures 1, ​,2A-B)2A-B) and 2× (Figure 2E) objectives was used. Filter sets used: enhanced green fluorescent protein/unconverted Kaede: SZX2-FGFPHQ (excitation, 460-480 nm; emission, 495-540 nm); dsRed/photoconverted Kaede/Neutral Red: SZX2-RFP2 (excitation, 540-580 nm; emission, 610 nm); and Kaede photoconversion: CFP CHR-U-N49001 (Chroma; excitation, 434-438 nm, emission 440-520 nm, exposure, 15-20 minutes). Original images were 1360 × 1024 RGB color (cropped).

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Figure 1

A 1.86-kb mpeg1 promoter fragment drives transient transgene expression in macrophages. (A) Transient mosaic transgene expression in embryos injected with tol2-flanked Tg(mpeg1:mCherry) (i) and Tg(mpeg1:EGFP) (ii) DNA constructs into Tg(lyz:EGFP) and Tg(lyz:dsRed) transgenic backgrounds, respectively. The experiment was conducted on Tg(lyz:EGFP) and Tg(lyz:dsRed) embryos to provide comparison and nonoverlap of expression with neutrophils. (B) Transgene expression in F0 adults injected with tol2-flanked Tg(mpeg1:mCherry) (i,iii) and Tg(mpeg1:EGFP) (ii) DNA constructs. (iii) Tg(mpeg1:mCherry) was introduced onto the Tg(mpx:EGFP) background to provide comparison and nonoverlap of expression with neutrophils. (C) Transient transgene expression resulting from delivery of a tol2-flanked Tg(mpeg1:Gal4-VP16) construct into Tg(UAS:Kaede) embryos results in expression of Kaede in dispersed cells (i) that migrate in response to wounding (tail transection) (ii-v). Time: hours post injury (hpi). (D) Delivery of tol2-flanked Tg(mpeg1:Gal4-VP16) into Tg(UAS:Kaede/lyz:dsRed) embryos allows comparison of macrophage (green, arrowheads) and neutrophil populations (red) migrating in response to wounding (tail transection). Time: hours post injury (hpi).

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Figure 2

Macrophage ontology, morphology, and behavior in stable mpeg1 transgenic zebrafish. (A) Unconverted Kaede expression (green) in dispersed macrophages in Tg(mpeg1:Gal4-VP16/UAS:Kaede) F1 embryos from 28 to 144 hpf. Images at 28, 50, and 120 hpf are composites assembled from photographs of the same embryo taken in 2 focal planes. (B) Loss of transgene expression occurs in young F1 and F2 Tg(mpeg1:Gal4-VP16/UAS:Kaede) adults (i,iv), demonstrated by the absence of dispersed fluorescent macrophages in the tail fin; compare with macrophages in the tail fins of Tg(mpeg1:mCherry) and Tg(mpeg1:EGFP) F0 animals in Figure 1B. However, the direct offspring of an outcross of the F1 adult (i) still shows strong embryonic transgene expression in dispersed macrophages (ii,iii). (I,iv) Arrowheads indicate autofluorescent iridophores. Bar represents 1 mm. (C) Dendritic morphology (arrowheads) of photoconverted Kaede (red) marked cells in Tg(mpeg1:Gal4-VP16/UAS:Kaede) F1 embryos. Bar represents 20 μm. (D) No overlap of fluorophore expression was observed between photoconverted Kaede and EGFP in F1 Tg(mpeg1:Gal4-VP16/UAS:Kaede/mpx:EGFP) compound transgenic embryos, demonstrating that the mpeg1 promoter drives expression in an entirely separate myeloid cell population to that of the mpx promoter (green represents neutrophils; and red, macrophages). Bar represents 50 μm. (E) Macrophage pinocytosis leads to accumulation of neutral red staining in vacuoles of unconverted Tg(mpeg1:Gal4-VP16/UAS:Kaede) positive cells (green) in the brain (arrowheads) of F1 embryos. Bar represents 50 μm. (F) Phagocytosis of heat-killed Penicillium marneffei spores (calcofluor-labeled, blue, arrowhead) by macrophages (red represents photoconverted Kaede) and neutrophils (green represents EGFP). Note phagocytosis of lower fungal spore by macrophage (bottom filled arrowhead) and migration of neutrophil with intracellular spore (open arrowhead). Stills from supplemental Video 1. Bar represents 50 μm. Time: minutes after infection.

For single photon confocal microscopy (Figure 2C-D), an Olympus FV1000-BX61WI upright multiphoton with an XLUMPlan F1 20×, water immersion, 0.95 NA objective was used. Excitation wavelengths used were 473 nm for EGFP and 559 nm for Kaede. Original image details were, for Figure 2Ci, xyz: 512 × 512 × 37 pixels, maximum intensity projection (cropped); for Figure 2Cii, xyz: 512 × 512 × 34 pixels, maximum intensity projection (cropped); for Figure 2D, (head) xyz: 512 × 512 × 113 pixels, maximum intensity projection; for Figure 2D, (ICM) xyz: 512 × 512 × 105 pixels, maximum intensity projection; and for Figure 2D (Tail) xyz: 512 × 512 × 140 pixels, maximum intensity projection.

Line-scanning confocal microscopy (Figures 2F, ​F,3A,3A, and Figure 4), was performed using a Zeiss LSM 5 Live inverted microscope with a Plan-Apochromat 20×, 0.8 NA objective. Wavelengths used were: 405 nm for Calcofluor; 488 nm for EGFP; and 561 nm for photoconverted Kaede. Original image details were, for Figure 2F, xyzt: 512 × 512 × 4 (pixels) × 100 (2 frames per minute) maximum intensity projection (cropped, deconvoluted); for Figure 3A, xyzt: 512 × 512 × 1 (pixels) × 1138 (2 frames per minute); for Figure 4A, xyzt: 512 × 512 × 1 (pixels) × 1138 (2 frames per minute; cropped); and for Figure 4B-D, xyzt: 512 × 512 × 1 (pixels) × 1800 (2 frames per minute) (cropped).

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Figure 3

Comparative descriptive and quantitative analysis of macrophage and neutrophil behavior after wounding. (A) Frames extracted from 11.5 hours of time lapse microscopy (supplemental Video 2) after tail transection in a Tg(mpeg1:Gal4-VP16/UAS:Kaede/mpx:EGFP) F1 embryo (red represents macrophages; and green, neutrophils). Bar represents 100 μm. (B-C) Cell migration paths for the first 6 neutrophils (B) and 6 macrophages (C) to arrive at the wound, followed for 18 hours after tail transection, extracted from a video of another wounded Tg(mpeg1:Gal4-VP16/UAS:Kaede/mpx:EGFP) F1 transgenic embryo. (D-H) Graphing of distance to wound edge for the same 6 neutrophils (D) and macrophages (E) as in panels B and C demonstrates an overall difference in migratory and dwelling behaviors. All macrophages migrate directly to the wound and remain near the wound edge for the remainder of the time course, whereas approximately 30% of neutrophils resume a roaming behavior from 3 to 6 hours after wounding. Insets i and ii for each panel present collected x/y movement vectors before (i) and after (ii) wound arrival. Strong directionality is demonstrated by both cell types before arrival, but macrophages move with a slower prearrival velocity (F) and with a stronger directionality reflected by their higher meandering index (direct path length/actual path length) (G). In-wound persistence index, the percentage of remaining time spent at the wound edge after arrival (H), demonstrates the propensity of macrophages to remain in the wound margin for long periods after their arrival, whereas neutrophils show a significantly larger spread of behaviors. Descriptive statistics: (B-E) Data for the same 6 cells of each type. (F-H) Data points are individual cells from 4 embryos imaged for 18 hours after wounding. (F,G) Bars represent mean plus or minus SD. Tests of significance: (F,G) t test, 2-tailed. (H) Mann-Whitney test, 2-tailed.

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Figure 4

Interactions between macrophages and neutrophils in vivo. (A) Interactions between macrophages (red represents photoconverted Kaede) and neutrophils (green represents EGFP) in Tg(mpeg1:Gal4-VP16/UAS:Kaede/mpx:EGFP) compound transgenic F1 embryos. Two examples of apoptotic neutrophils being phagocytosed by macrophages at the wound margin. The first occurs at 340 minutes by macrophage 1 (Mϕ-1) at the top of frame followed by the second at 348 minutes by Mϕ-2. Loss of green neutrophil fluorescence is evident in the Mϕ-1 after 76 minutes and within Mϕ-2 occurs in 45 minutes. Stills from supplemental Video 3. Bar represents 10 μm. (B) Demonstration that the loss of cytoplasmic neutrophil fluorescence is dependent on macrophage phagocytosis. At 568 to 571 minutes, partial phagocytosis of a segment of a neutrophil. At 572.5 to 634 minutes, loss of fluorescence in the phagocytosed fragment occurs after approximately 6 hours (dotted circle represents region of lost EGFP fluorescence). Providing an internal control for this process, an unphagocytosed still-fluorescing neutrophil fragment remains stationary throughout the first phagocytic phase, until it is subsequently engulfed by the same macrophage at 727.5 minutes, with loss of its EGFP fluorescence at 736.5 minutes. Stills from supplemental Video 4. Bar represents 10 μm. In both panels, white arrowheads and yellow arrowheads indicate unphagocyted and phagocytosed states, respectively, of neutrophil corpse. (C-D) Two examples of cytoplasmic transfer from live neutrophils (green represents EGFP) to macrophages (red represents photoconverted Kaede). (C) A neutrophil/macrophage interaction near a wound margin results in a cytoplasmic fragment from a live neutrophil transferring to within a macrophage. (Top panel) Merged images. (Bottom panel) EGFP channel only. Dashed outlines indicate position of macrophage (blue) and neutrophil/fragment. Stills extracted from supplemental Video 5. Bar represents 20 μm. (D) A neutrophil/macrophage interaction in the trunk of the embryo. Merged fluorescence and bright-field images clearly demonstrate interaction resulting in cytoplasm transfer from neutrophil to macrophage. Stills from supplemental Video 6. Bar represents 20 μm. In both examples, white arrowheads and yellow arrowheads indicate unphagocytosed and phagocytosed states, respectively, of the transferring neutrophil cytoplasmic fragment. Neutrophil viability throughout the process is demonstrated by its subsequent migration out of the field of view (direction of white arrow in subpanels iv and v).

Wide-field microscopy for cell-tracking studies (Figure 3) was performed using a Nikon Ti-E inverted microscope with a Plan Fluor 10×, 0.3 NA objective. Wavelength filters used were FITC (Nikon; excitation 465-495 nm; emission, 515-565) for EGFP and TRITC (Nikon; excitation 515-565 nm; emission 550-660) for photoconverted Kaede. Original image details were Figure 3B-H, xyzt: 512 × 512 × 4 (pixels, 2 × binning) × 1080, 1 frame per minute). Deconvolution and maximum intensity projection were performed before tracking analysis.

Images were processed in Adobe Creative Suite CS4 and ImageJ, Version 1.4q, programs for presentation. Quantitative leukocyte behavior measurements were extracted using Metamorph (Series 7.5). The “meandering index” is as previously defined (linear distance between start and finish points divided by actual path length).³³ The “in-wound persistence index” was defined as the percentage of remaining time in a time lapse that a cell spent at the wound edge after arrival.

The mpeg1 promoter drives transgene expression in macrophages

Several mpeg1-driven, tol2-flanked transgene constructs were built for recapitulating mpeg1 expression in live animals: Tg(mpeg1:EGFP); Tg(mpeg1:mCherry); and Tg(mpeg1:Gal4-VP16). In F0-microinjected embryos, transgene expression was observed in a dispersed population of mobile, putative myeloid cells (Figure 1A-C). Inflammation assays using F0 Tg(mpeg1:Gal4-VP16/UAS:Kaede) embryos demonstrated Kaede-expressing cells migrating in response to wounding (Figure 1C-D), a characteristic leukocyte behavior. Encouragingly, in F0s, no overlap of fluorophore expression was observed between mpeg1-driven transgenes and characterized neutrophil-specific Tg(mpx:EGFP) and lyz-driven transgenes (Figure 1A-B,D), although these F0 embryos were potentially mosaic.

Stable germline transgenic Tg(mpeg1:Gal4-VP16) zebrafish were generated to provide opportunity for this promoter to drive a range of effector gene functionalities (Figure 2). Initial transgenesis was performed on a Tg(UAS:Kaede) background. mpeg1-transgene-marked cells were present into early larval stages of F0, F1 (Figure 2A), and F2 embryos (Figure 2Bii-iii). However, transgene expressing cells were observed only rarely in Tg(mpeg1:Gal4-VP16/UAS:Kaede) F1 and F2 adults (Figure 2Bi,iv). In contrast, Tg(mpeg1:mCherry) and Tg(mpeg1:EGFP) F0 adults showed strong transgene expression (Figure 1B).

To assess overlap between expression of the new mpeg1-driven transgene and a standard neutrophil marker in nonmosaic F1s, F0 founders were crossed into the Tg(mpx:EGFP) line³ and compound transgenic Tg(mpeg1:Gal4-VP16/UAS:Kaede/mpx:EGFP) F1 embryos were imaged after photoconversion of Kaede. No overlap at all was observed between cell populations in the embryos at 2, 3, or 7 dpf (number of scored cells expressing mpeg1/mpx/both: at 2 dpf, 50/125/0; at 3 dpf, 60/306/0; at 7 dpf, 222/401/0; scores collected from multiple fields in ≥ 2 embryos), establishing that in this line the mpeg1 promoter drives expression exclusively in a nonneutrophil cell population (Figure 2D).

The fluorescent cells in Tg(mpeg1:Gal4-VP16/UAS:Kaede) embryos had several defining morphologic and functional attributes characteristic of macrophage-lineage cells. They were large cells with extensive cytoplasmic extensions (50-100 μm including branches, Figure 2C). Incubation with neutral red led to accumulation of stain via pinocytosis (Figure 2E), as previously described for primitive zebrafish macrophages.³¹ The cells were phagocytic: injection of calcofluor-stained fungal conidia into the trunk led to migration of marked cells to the wound site and phagocytosis of microbes (Figure 2F; supplemental Video 1).

During inflammation, macrophages migrate with different kinetics to neutrophils

F1 Tg(mpeg1:Gal4-VP16/UAS:Kaede/mpx:EGFP) transgenic embryos were wounded and imaged, using photoconverted Kaede (red) to discriminate macrophages from EGFP-expressing (green) neutrophils (Figure 3, supplemental Video 2).

During the arrival phase, both neutrophils and macrophages migrated toward the site of injury with strong directionality, demonstrated by their tracks (Figure 3B-C) and arrival movement vectors (Figure 3Di,Ei) during migration. Compared with the swift initial neutrophil migration toward the wound averaging 15 μm/minute, macrophages were significantly tardier, extending more pseudopodia and migrating more slowly (6 μm/minute; Figure 3F; supplemental Video 2). Macrophages took a more direct route to the wound margin, reflected in a significantly higher meandering index (0.55) than neutrophils (0.4; Figure 3G). Macrophages arriving at the wound in the first 24 hours remained for as long as 4 days after wounding, a behavior proven by localized photoconversion of Kaede at the wound site and monitoring cell location thereafter (supplemental Figure 2B-C), substantially extending previous observations.^36,37 Additional macrophages continued to arrive until at least 48 hours after wounding (supplemental Figure 2C). In contrast, neutrophils discontinued directional migration toward the wound after 6 hours (Figure 3A; supplemental Video 2).

Neutrophils and macrophages also behaved differently after arrival at the wound. During 18- hour time lapses, approximately 30% of neutrophils that arrived at the wound margin resumed a roaming behavior from 3 to 6 hours after wounding (Figure 3D,H), whereas more than 90% macrophages dwelt persistently at the wound margin after their arrival (Figure 3E,H).

These data demonstrate the utility of this new transgenic zebrafish line, in combination with other transgenes, to observe, compare, and quantify distinctive macrophage behaviors.

The authors thank Ethan Scott, Herwig Baier, Stephen Renshaw, Chris Hall, and Phil Crosier for transgenic lines; Cameron Nowell and Kelly Rogers for assistance with imaging; Harshini Weerasinghe and Sony Varma for technical assistance; Mark Greer, Kelly Turner, and Prue Chamberlain for animal care in the aquarium; Ben Kile, Joan Heath, and Ben Croker for discussions; Steve Jane, David Curtis, and the Royal Melbourne Hospital Bone Marrow Research Laboratory for support. Microscopy used instruments in the Center for Advanced Microscopy (Ludwig Institute for Cancer Research) and the Australian Cancer Research Foundation Center for Therapeutic Target Discovery.

This work was supported by the National Institutes of Health (grant R01 HL079545) and the National Health and Medical Research Council (grant 637394; G.J.L.). F.E. was supported by an Australian Postgraduate Award and Walter and Eliza Hall Institute of Medical Research Edith Moffatt Scholarship. Walter and Eliza Hall Institute of Medical Research receives infrastructure support from the Commonwealth National Health and Medical Research Council Independent Research Institutes Infrastructure Support Scheme (361646) and a Victorian State Government Operational Infrastructure Support Scheme grant.