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First published online September 19, 2007
doi: 10.1242/10.1242/jcs.009159

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Live imaging of chronic inflammation caused by mutation of zebrafish Hai1

¹ Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USA
² Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA

View larger version (94K): [in this window] [in a new window]   Fig. 1. The hi2217 mutant exhibits a chronic inflammation phenotype and reduced expression of Hai1. (A) Genomic map of hi2217 insertion; exons are depicted as numbered boxes, and the start of the Hai1 (HAI-1) open reading frame (blue) is indicated (ATG); each section equals 500 bp. (B-F') Images of hi2217 embryos; B-G are wild-type siblings, B'-G' are mutants. (B,B') In situ hybridization of zebrafish MPO at 2 dpf, arrows indicate the position of the intermediate cell mass (ICM), the location of neutrophil development in zebrafish embryos. (C,C') Oblique coherent contrast (OCC) images of live hi2217 embryos at 26 hpf, arrows indicate clusters of rounded cells on the yolk sac (ys) and yolk sac extension (yse) of the mutant. (D-E') Hematoxylin and Eosin-stained transverse sections of hi2217 embryos at 2 dpf, at the level of the yolk sac extension (yse); s, somites; n, notochord. D,D' are full views; E,E' are magnified views of the boxed areas in D,D'. (F-G') In situ hybridization of Hai1 at 30 hpf. (F,F') Whole-mount and (G,G') frozen sections at the level of the yolk sac, which is indicated by dashed lines in F,F'; arrowheads indicate the epidermis. Whole embryos are oriented anterior to the left, dorsal side up; sections oriented dorsal side up. Bars, 200 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 2. Onset of neutrophilic inflammation in hi2217 mutants at 36 hpf. Tailfin views of hi2217 embryos fixed at 36 hpf; A-C are wild-type siblings, A'-C' are mutants. (A,A') OCC images and corresponding images of zMPO immunolabeling (B,B') and TUNEL (C,C') from the same embryo; arrows indicate the same group of rounded cells in A'-C'. Embryos are oriented anterior to the left, dorsal side up. Bars, 100 µm.

View larger version (138K): [in this window] [in a new window]   Fig. 3. hi2217 mutant phenotypes at 48 hpf. Tailfin views of hi2217 embryos fixed at 48 hpf; A-E are wild-type siblings, A'-E' are mutants. (A,A') OCC images and corresponding images of zMPO immunolabeling (B,B') of the same embryo; (C,C') L-Plastin immunolabeling in different embryos; (D,D') BrdU immunolabeling; (E,E') Pan-Cadherin immunolabeling. Embryos are oriented anterior to the left, dorsal side up. Bars, 100 µm.

View larger version (140K): [in this window] [in a new window]   Fig. 4. hi2217 mutant epithelial phenotypes at 38 hpf are not mediated by leukocytes. Tailfin views of hi2217 embryos pulse-labeled with BrdU and fixed at 38 hpf; A-F are wild-type siblings, A'-F' are mutants. (A,A',D,D') OCC images and corresponding images of zMPO immunolabeling (B,B',E,E') and BrdU immunolabeling (C,C',F,F') of uninjected (A-C,A'-C') and pu.1 MO-injected (D-F,D'-F') hi2217 embryos. Embryos are oriented anterior to the left, dorsal side up. Bar, 100 µm.

View larger version (94K): [in this window] [in a new window]   Fig. 5. Injection of hai1 MO into wild-type embryos phenocopies the hi2217 mutant. Shown are images of wild-type embryos at 24 hpf (A,A') and 48 hpf (B-E) following injection of a standard control MO (A), hai1 5-base mismatch MO (B,C) or hai1 MO (A'-C'). (A,A') Live OCC images; (B,B') OCC images and (C,C') corresponding zMPO immunolabeling in fixed embryos. (D,E) Partial knock-down by co-injection of hai1 MO and TMR-dextran (to trace injected cells) into 8- to 16-cell wild-type embryos. (D) OCC overlaid with red fluorescence from TMR-dextran, indicating cells that contain the MO, (E) Red fluorescence (TMR-dextran) overlaid onto green zMPO immunolabel. Note the otherwise wild-type morphology of the tailfin (compare to B,B') with localized areas of rounded cells (arrows) that overlap with red fluorescent cells, to which neutrophils (green) are recruited. Embryos are oriented anterior to the left, dorsal side up. Bars, 100 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 6. MO-mediated knock-down of matriptase 1 rescues hi2217 mutant. (A) In situ hybridization of zebrafish matriptase 1 in wild-type embryo at 30 hpf; inset shows an embryo labeled with a sense probe from the same plasmid template. (B-G) Live images of hi2217 embryos injected with either matriptase 1 MO1 (B,C,D,F) a standard control MO (E) or a 5-base mismatch matriptase 1 MO1 (G) at 30 hpf (B-E) or 48 hpf (F,G). (H) Quantification of morphological data at 30 and 48 hpf; the number of embryos exhibiting a mutant (mut) morphology (as in D,E,G) were counted for each condition and divided by the total number of embryos to give the percentage that exhibit the mutant phenotype (%). Note that any instance of rounded cells (e.g. that seen in D, even with an otherwise wild-type morphology) was counted as a mutant phenotype. (I,J) MPO activity assay of embryos fixed at 48 hpf following injection with matriptase 1 MO1 (I) or 5-base mismatch matriptase 1 MO1 (J). (K,L) Partial phenocopy by injection of a lesser volume of matriptase 1 MO. OCC (K) and corresponding MPO immunolabeling (L) of an hi2217 embryo injected with matriptase 1 MO2 at a concentration less than required for rescue (see Materials and Methods), note the recruitment of neutrophils to localized areas of rounded cells (arrows in K). Note that the similar injection of 5-base mismatch matriptase 1 MO1 into other embryos resulted in 25% showing a `full' mutant phenotype similar to that seen in G and J (data not shown). Embryos are oriented anterior to the left, dorsal side up (A-C,F-L) or anterior up, dorsal side to the right (D,E). Bars, 100 µm.

View larger version (105K): [in this window] [in a new window]   Fig. 7. Migratory behavior of neutrophils in the hi2217;MPO:GFP fish. (A) Single frame from time-lapse movie (Movie 1 in supplementary material) of hi2217;MPO:GFP embryo tailfin at 2 dpf, fluorescence overlaid onto DIC images. Three individual tracks of GFP-positive neutrophils are shown, with the start of each track indicated with an arrow. Dashed boxes indicate selected segments of tracks, depicted in B and C. Bar, 100 µm. (B,C) Time-lapse sequences of tracks from A, with times indicated at the top of each image in hours:minutes:seconds; the side of each image box equals 50 µm. Large arrows indicate the direction of motility, small arrowheads mark pseudopodia. Note how mutant neutrophils frequently stop and extend pseudopodia. (D) Higher magnification (box side, 25 µm) sequence of a single neutrophil; otherwise same as B,C. Note how several pseudopodia are extended over a 2-minute period. (E) Plot of average velocity against percentage of time stopped (percentage of distance data points less than 0.75 µm) for full-length mutant tracks; each point indicates the parameters for an individual cell, dashed lines indicate the average values (in parentheses) for each data set. (F-I) Analysis of hi2217 mutant neutrophil tracks in 15-minute windows. Single frames from time-lapse movies of hi2217;MPO:GFP embryo tailfins at 2 dpf are shown; fluorescence overlaid onto DIC images. Individual tracks of GFP-positive neutrophils are shown, with the start of each track indicated by an arrow; bars, 100 µm. Next to each track image is a plot of directional persistence (D/T, blue lines) and velocity (µm/minute, red lines) over 15-minute time intervals (x axis) for the length of each track. D/T values are multiplied by 10 to use the same numerical y axis as velocity; therefore D/T values above 7 (dashed lines) indicate directional migration.

View larger version (40K): [in this window] [in a new window]   Fig. 8. Neutrophils in hi2217 mutant embryos display random migration as compared to wild-type neutrophils responding to a wound, but retain the ability to respond directionally to acute injury. (A,B) `Mutant' data are derived from the first 12 minutes of each hi2217 mutant track, `WT Wound' and `Mutant Wound' data are derived from tracks of neutrophils in response to tailfin wounds in mutants and wild-type siblings, respectively. (A) Table summarizing cell tracking data, including the number of cells tracked (#), average velocity (V, µm/minute) and average directional persistence (D/T); standard deviations are listed in parentheses. (B) Plot of average velocity (µm/minute, y axis) against directionality (D/T, x axis). Each point indicates the parameters for an individual cell; blue points are homozygous mutants, red points are wild-type siblings, yellow points are wounded mutants; dashed lines indicate the average values for each data set. (C) Plot of average mean displacement (µm, y axis) against square root of time interval (minute1/2, x axis) for the first 8.5 minute of each track; error bars indicate the standard error of the mean. Blue points are homozygous mutants, red points are derived from tracks of neutrophils in response to tailfin wounds in wild-type siblings. (D,E) Mutant neutrophil response to tissue injury. (D) A homozygous mutant hi2217;MPO:GFP embryo at 2 dpf was briefly analyzed by time-lapse microscopy: a single neutrophil (arrows indicate the same cell in each frame) is tracked to an area of cell rounding (yellow brackets) in the ventral fin; indicated times are from the start of the movie. (E) The same embryo was then wounded (indicated with *) to the left of the bracketed area, and neutrophils (arrows or arrowheads mark two individual cells in each frame) were observed to migrate to the wound, with several neutrophils at the wound by 7.5 minutes (indicated times are post-wound). Tracks for four individual neutrophils are overlaid in the last frame. Bar, 50 µm.

View larger version (37K): [in this window] [in a new window]   Fig. 9. NS-398 treatment causes neutrophil arrest in hi2217;MPO:GFP embryos. (A) Mutant hi2217;MPO:GFP embryo tailfin following treatment for 1 hour in 250 µM NS-398. Fluorescence image of the first frame from Movie 3 in supplementary material overlaid onto corresponding OCC image; dashed line indicates the location of the vasculature. (B) Cell tracks of neutrophils from embryo in A, kept in the presence of NS-398. (C) Tracks from same embryo as in A and B, 1 hour after washing out the drug. Average displacements for each set of tracks are indicated in the lower left corner of B and C.