Factor associated with neutral sphingomyelinase activity mediates navigational capacity of leukocytes responding to wounds and infection: live imaging studies in zebrafish larvae

Abstract

Factor associated with neutral sphingomyelinase activity (FAN) is an adaptor protein that specifically binds to the p55 receptor for TNF (TNF-RI). Our previous investigations demonstrated that FAN plays a role in TNF-induced actin reorganization by connecting the plasma membrane with actin cytoskeleton, suggesting that FAN may impact on cellular motility in response to TNF and in the context of immune inflammatory conditions. In this study, we used the translucent zebrafish larvae for in vivo analysis of leukocyte migration after morpholino knockdown of FAN. FAN-deficient zebrafish leukocytes were impaired in their migration toward tail fin wounds, leading to a reduced number of cells reaching the wound. Furthermore, FAN-deficient leukocytes show an impaired response to bacterial infections, suggesting that FAN is generally required for the directed chemotactic response of immune cells independent of the nature of the stimulus. Cell-tracking analysis up to 3 h after injury revealed that the reduced number of leukocytes is not due to a reduction in random motility or speed of movement. Leukocytes from FAN-deficient embryos protrude pseudopodia in all directions instead of having one clear leading edge. Our results suggest that FAN-deficient leukocytes exhibit an impaired navigational capacity, leading to a disrupted chemotactic response.

FIGURE 1.

(A) Schematic drawing of the primary structure of zebrafish FAN. The PH, BEACH, and WD40 domains are shown in yellow, dark green, and red, respectively. (B) RT-PCR analysis of FAN during the early stages of development. mRNA was isolated from embryos at the indicated time points; β-actin was used as a positive control. FAN mRNA is already maternally provided and strongly expressed during the first 6 d of development. (C) RT-PCR to monitor the effect of the injected FAN antisense morpholinos. Injection of the single morpholinos (I1E2, E2I2) and combined injection (DI) abolishes the wild-type transcript. The combined injection of both morpholinos leads to the generation of two shorter splice variants. Sequence analysis of the two generated transcripts revealed a deletion of exons 2 and 3 and parts of exon 4 within the shorter transcript. Single injections were performed with a morpholino concentration of 0.3 mmol. Double injections were performed with a morpholino concentration of 0.3 mmol and 0.6 mmol. (D) Whole-mount in situ hybridization for l-plastin to visualize early leukocytes at 32 h postfertilization (hpf). For the in situ hybridization digoxygenin-labeled probes were used. The embryos were incubated with digoxygenin-labeled anti-sense RNA probes. The hybridized probes were detected immunochemically, by means of alkaline phosphatase-conjugated anti-digoxygenin Fab fragments, whereby the enzymatic conversion of specific substrates resulted in the production of colored precipitates. Small pictures on the right side are zoom-in areas for the head and the tail. No difference in number and distribution between wild-type and FAN knockdown embryos could be detected.

FIGURE 2.

(A) Tail fin of a zebrafish embryo before laser injury. Wild-type, control, and FAN-MO–injected embryo 1.5 h after laser injury. The white arrow indicates the position of injury. The dashed line indicates the area in which leukocytes were counted after 1.5 h. (B) Statistical illustration of the number of leukocytes recruited to wild-type, control, and morphant wounds (Wt, n = 19, Ctrl, n = 20, MO, n = 35; ***p value Wt/MO <0.000004, ***p value Ctrl/MO <0.00005). (C and D) Sudan black staining and fluorescence analysis in Pu1-GAL4-UAS-GFP embryos. Embryos were injured with a scalpel. Embryos were fixed 2 h after tail fin injury. Green cells represent the total number of leukocytes that were recruited to the wound; neutrophils were stained with Sudan black (arrowheads). (E) Statistical illustration of the total number of leukocytes and the number of neutrophils recruited to control and morphant wounds after Sudan black staining and fluorescence analysis (Ctrl, n = 16, MO, n = 15; **p value Ctrl/MO [Pu1] <0.005, *p value Ctrl/MO [Sudan black] <0.03). (F) Statistical illustration of the total number of leukocytes and the number of neutrophils recruited to control and morphant wounds in double-transgenic embryos (PU1-Gal4-UAS-RFP; MPX-GFP) (Ctrl, n = 5, MO, n = 5; ***p value Ctrl/MO [PU1] <0.001, ***p value Ctrl/MO [MPX] <0.001).

FIGURE 3.

(A) Cell tracking from fluorescence movies up to 3 h after injury at the tail fin from 2-dpf embryos treated with control morpholino or FAN morpholino. A transgenic fishline (PU1-Gal4 UAS-GFP) was used for our experiments that has green fluorescent leukocytes. ×40 water objective. (B) Statistical illustration of the speed of leukocytes recruited to wounds in wild-type, control, and morphant embryos (Wt, n = 18, Ctrl, n = 18, MO, n = 26; p value Wt/MO <0.1 n.s., *p value Ctrl/MO <0.02). (C) Statistical illustration of the straightness of leukocytes recruited to wounds in wild-type, control, and morphant embryos (Wt, n = 18, Ctrl, n = 18, MO, n = 26; ***p value Wt/MO <0.000003, ***p value Ctrl/MO <0.00002).

FIGURE 4.

(A) Overview of cell tracking of a wild-type embryo. The white circle indicates the site of injury. (B) Detailed cell tracking of one leukocyte. The macrophage moves directly toward the wound and reaches it after 24 min. After reaching the wound, it stays there and does not move away from it. (C) Overview of cell tracking of a FAN morphant embryo. The white circle indicates the site of injury. (D) Detailed cell tracking of one leukocyte. The leukocyte does not move directly toward the wound, but performs protrusions in every direction before starting to move after 55 min. After 70 min, the macrophage reaches the wound, but directly starts to move away again. Single leukocytes were manually colored in red and blue by using ImageJ software. ×40 water objective.

FIGURE 5.

(A) A typical shape of control leukocytes. Leukocytes have one clear leading edge and a retracting part without pseudopodia. Arrow indicates the direction of move. Arrowheads indicate pseudopodia. (B) A typical shape of morphant leukocytes. Leukocytes extend pseudopodia in all directions without having a clear leading edge. Arrow indicates the direction of move. Arrowheads indicate pseudopodia. (C) Graphic illustration of the proportion of right choices (green) versus wrong choices (red) of pseudopodia made by control and wild-type versus morphant leukocytes (number of leukocytes: Wt, n = 10, Ctrl, = 10, MO, = 10; ***p value Wt/MO <0.0002, ***p value Ctrl/MO <0.0004).

FIGURE 6.

(A) 3-dpf zebrafish larvae. The black rectangle marks the optic tectum, which is shown in a higher magnification (original magnification ×40) on top. (B and C) Fluorescent images of the optic tectum infected with dsRED-labeled E. coli in control (B) and FAN morphant embryos (C). (B) and (C) show the green fluorescent leukocytes of the transgenic fishline Pu1-Gal4 UAS-GFP and the dsRed-expressing E. coli injected into the otic placode. ×40 water objective. (D) Statistical illustration numbers of leukocytes recruited to wild-type, control, and morphant otic placodes postinfection with red fluorescent E. coli (Wt, n = 5, Ctrl, n = 5, MO, n = 5; **p value Wt/MO <0.003, *p value Ctrl/MO <0.01).