Distinct mechanisms regulate hemocyte chemotaxis during development and wound healing in Drosophila melanogaster

Abstract

Drosophila melanogaster hemocytes are highly motile macrophage-like cells that undergo a stereotypic pattern of migration to populate the whole embryo by late embryogenesis. We demonstrate that the migratory patterns of hemocytes at the embryonic ventral midline are orchestrated by chemotactic signals from the PDGF/VEGF ligands Pvf2 and -3 and that these directed migrations occur independently of phosphoinositide 3-kinase (PI3K) signaling. In contrast, using both laser ablation and a novel wounding assay that allows localized treatment with inhibitory drugs, we show that PI3K is essential for hemocyte chemotaxis toward wounds and that Pvf signals and PDGF/VEGF receptor expression are not required for this rapid chemotactic response. Our results demonstrate that at least two separate mechanisms operate in D. melanogaster embryos to direct hemocyte migration and show that although PI3K is crucial for hemocytes to sense a chemotactic gradient from a wound, it is not required to sense the growth factor signals that coordinate their developmental migrations along the ventral midline during embryogenesis.

Figure 1.

Developmental dispersal of hemocytes at the ventral midline. (A) Ventral aspect of a pxnGAL4 UAS-GFP embryo at stage 12, showing hemocytes as they migrate along the ventral midline from anterior to posterior. (B) High-magnification detail of hemocytes at this stage reveals that cells are clustered together and exhibit only a few protrusions (arrow). (C) Ventral view of pxnGAL4 UAS-GFP embryos at stage 16. (D) High magnification of the same embryo reveals that these ventrally placed hemocytes are much larger than early hemocytes (compare with B), exhibiting impressive filopodia and lamellipodia. (E) Stills from a time-lapse video show hemocyte dispersal between Stages 14.5 and 15.5 of development. In all images, anterior is up and posterior is down. At the beginning of this video, the majority of hemocytes are positioned in a single line along the midline (i). Over the next 150 min of development, individual hemocytes leave the midline and migrate laterally to form the two more laterally placed populations seen at stage 16 (v). (F) Superimposing the migratory paths of all hemocytes that leave the midline shows this process to conform to a pattern with hemocytes migrating away from the midline at a 90° angle and leaving from spatially reiterated exit points (i and v, colored circles). Throughout this movement. midline hemocytes favor these exit points and often clump together at these locations (iii, arrows). A hemocyte can be seen at each of these locations once the migration is complete (v). Individual hemocytes are labeled with colored numbers as they migrate laterally, with the color corresponding to the exit point from which they left the midline. Elapsed time in minutes is indicated in the lower right corner. Bars, 20 μm.

Figure 2.

Quantification of directionality, velocity, and polarity of hemocytes during lateral migration. (A) A typical path of a hemocyte performing lateral migration from the midline. Three distinct phases (a, b, and c) can be identified. Before starting the lateral migration, while in the midline, the hemocyte moves between adjacent exit points before adopting a more random path with constant changes in direction (blue path). This is followed by the lateral migration (red path) displaying high directionality (calculated by applying a ratio between the real distance traversed by the hemocytes and the shortest possible distance shown by the broken white line). On arrival at its destination, the hemocyte returns to a more random directional behavior (yellow path). (B) The same path represented by dots marking the hemocyte position at each time point shows the corresponding change in velocity of the hemocyte during lateral migration (distance between dots corresponds to velocity). (C) Confocal image of ventral aspect of a srpHemoGAL4 UASGFP embryo during lateral hemocyte migration. (D) Stills from a time-lapse video showing lateral migration of the hemocyte (C, box 1). The hemocyte has been artificially divided into quadrants (1, 2, 3, and 4) corresponding to posterior medial, anterior medial, anterior lateral, and posterior medial, respectively. The outline of the cell has been traced to allow measurement of protrusive area. As the hemocyte migrates laterally from the midline, it extends a large membrane ruffle from its leading edge and displays little or no protrusions on its medial side. (E) Graph showing values for protrusive area in each quadrant plotted against time shows the difference between protrusive area at the lateral side of the cell (pink and purple lines) when compared with the medial side (blue and yellow lines). (F) Stills from a time-lapse video showing protrusion dynamics of hemocyte (C, box 2). This hemocyte remains in the midline and displays a more random distribution of membrane ruffles, with protrusions rarely remaining in the same cell quandrant for >2 min. Elapsed time is indicated in the corner. Bars: (C) 20 μm; (D and F) 50 μm.

Figure 3.

Embryonic expression of Pvf genes. In situ hybridization showing the expression patterns of Pvf1 (A–E), Pvf2 (F–J), and Pvf3 (K–O) during embryonic development. (A–D) Pvf1 is not expressed within the ventral midline at any point during embryogenesis. However, strong expression was detected in the tracheal cells, caudal ectoderm, malpighian tubules, and a posterior ectodermal ring as previously reported (Cho et al., 2002). (F–I) No Pvf2 expression was detected in the ventral midline at stage 10 and was only observed from stage 12 onward. Expression appears strongest at stage 14 and, over the next 2 h of development, RNA levels decrease in a wave from anterior to posterior such that by stage 15 the ligand is expressed in small, segmentally reiterated points along the posterior region of the ventral nerve cord. (K–M) Pvf3 is expressed along the ventral midline at stage 10, when the germ band is fully extended. This expression subsequently decreases and falls to almost undetectable levels by stage 14. (J) High expression levels of Pvf2 were also detected in the dorsal vessel at stage 14. No such staining was observed using probes against Pvf1 (E) or Pvf3 (O). Bar, 50 μm.

Figure 4.

Pvf2 and -3 act redundantly as hemocyte chemoattractants in the CNS, and Pvf2 acts alone in the dorsal vessel. (A and B) srpHemoGAL4UASGFP embryos stained with anti-armadillo (red) and anti-GFP (green) antibodies to visualize wild-type hemocyte migration patterns. (A) Dorsal aspect of an embryo at stage 14 shows hemocytes migrating posteriorly, along the dorsal vessel (arrows). (B) Ventral aspect of a stage 16 embryo shows hemocytes occupying three parallel lines running anterior to posterior along the embryonic ventral midline. (C and D) pvr¹ mutant embryos expressing UAS-p35 and -GFP under the control of the hemocyte-specific srpHemoGAL4 driver (Bruckner et al., 2004). (C) Dorsal aspect of a stage 14 embryo shows that although hemocytes are able to migrate away from their origin in the head, no hemocytes are found along the dorsal vessel (arrows) in these mutant embryos. (D) Ventral aspect of a stage 16 embryo shows that despite expression of p35, pvr¹ mutant hemocytes are completely absent from the ventral midline and only a small number can be seen in the more lateral positions (arrow). (E and F) pvf2 mutant embryos (vegf27Cb^c6947) double stained with anti-armadillo (red) and anti-croquemort (green). These mutants display a complete absence of hemocytes at the dorsal vessel (E, arrows) but only a slight reduction in hemocyte number at the ventral midline (F). (G–L) srpHemoGAL4UASGFP embryos injected with dsRNA against Pvf2 (G and H), Pvf3 (I and J), and Pvf2 and -3 together (K and L). Embryos have been stained using the same antibodies used in A and B. Embryos injected with Pvf2 dsRNA show the same phenotype as the pvf2 mutant (G and H). Injection of Pvf3 dsRNA had little effect on hemocyte migration (I and J); however, simultaneous silencing of both Pvf2 and -3 prevented hemocytes migrating along both the dorsal vessel (K) and the ventral nerve cord (L), mimicking the phenotype observed in rescued UAS-p35–expressing pvr¹ mutants (compare L and D). Bar, 50 μm.

Figure 5.

Lateral hemocyte migration at the ventral midline requires a down-regulation in Pvf2. (A–D) Embryos double stained with antibodies against armadillo (red) and croquemort (green). (A) Ventral aspect of a wild-type stage 15 embryo shows the normal pattern of hemocyte distribution with many cells located in lateral positions (arrows), having migrated away from the midline (asterisks). (B) Ventral aspect of a late stage 16 wild-type embryo. Hemocytes can clearly be seen in the characteristic three lines running anterior to posterior along the embryo. (C) Stage 15 embryo in which Pvf2 is being overexpressed in the ventral midline using the driver simGAL4. Antibody staining shows that the normal hemocyte distribution has been drastically disrupted, with only a small number of hemocytes found in lateral positions (arrows) and the majority remaining in clumps along the midline (asterisks; compare with A). (D) By late stage 16, these simGAL4 UASPvf2 embryos show normal lateral hemocyte distribution (arrows) and no trace of the delay in lateral migration can be seen. Bars, 50 μm.

Figure 6.

Bead application leads to actin-dependent hemocyte chemotaxis. (A) Differential interference contrast image of a wound made to a UAS-p35–expressing pvr¹ mutant. (B) Fluorescent image of the same wound as in C shows normal hemocyte numbers at the wound 1 h after laser ablation. Broken line indicates wound margin. (C) Implanting a bead ∼30 μm in diameter into an embryo creates an epithelial wound of the same diameter as seen in an E-cadherin–GFP–expressing embryo. (D) When untreated beads are administered to a pxnGAL4 UAS-GFP embryo, a rapid accumulation of hemocytes can be seen at the wound site, which surround the bead within 30 min. (E) Hemocytes surrounding a bead presoaked in DMSO exhibit large lamellipodia (arrows) as they actively try to engulf the bead. (F) Bead presoaked in Latrunculin B in DMSO shows a dramatic reduction in number of hemocytes around the bead when compared with the control, and those cells near the bead exhibit no lamellipodia (though some rudimentary protrusions can still be seen). Hemocytes lying 50 μm away from the bead, however, are able to form lamellipodia (arrow) because of a drop off in drug concentration. (G) Similarly, implantation of a bead soaked in Cytochalasin D in DMSO blocks hemocyte recruitment, and those cells exposed to the drug fail to form large actin protrusions and are consequently unable to migrate (see Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200508161/DC1). Bars: (A) 50 μm; (B–G) 20 μm.

Figure 7.

Hemocyte developmental dispersal does not require PI3K. E-cadherin–GFP; crqGAL4 UAS-GFP embryos reveal normal hemocyte distribution at stages 12 (A), 14 (C), and 16 (E) of development. (B, D, and F) Images showing developmental distribution of dominant-negative PI3K (Dp110^D954A)–expressing hemocytes. The migrations of the dominant-negative–expressing hemocytes are indistinguishable from those of the wild type. Bar, 50 μm.

Figure 8.

PI3K is required for hemocyte chemotaxis toward wounds. (A) Wound made to a pxnGAL4 UAS-GFP embryo and double stained using antibodies against armadillo (red) and GFP (green) that highlights the homocytes (arrows). (B) The same antibody staining on a wounded embryo expressing Dp110^D954A, specifically in the hemocytes, reveals a dramatic decrease in the number of hemocytes at the wound site (arrow). (C) Low-magnification image showing a pxnGAL4 UAS-GFP embryo 1 h after bead implantation. The bead (arrow) is surrounded by activated hemocytes. (D) In contrast, when a bead (arrow) is implanted in an embryo expressing Dp110^D954A, specifically in the hemocytes, these mutant cells fail to chemotax toward the wound site and the bead remains undetected by the hemocytes 1 h after implantation. (E) To verify this result, we implanted two beads into a pxnGAL4 UAS-GFP embryo; one bead was untreated (blue) and the other was presoaked in the PI3K inhibitor LY294002 (red). (F) 1 h after bead implantation, the untreated bead (arrowhead) is surrounded by hemocytes, whereas these cells have completely failed to chemotax toward the bead (arrow) soaked in PI3K inhibitor. (G) Graph showing mean numbers of hemocytes surrounding implanted beads in wild type, Dp110^D954A-expressing embryos, and drug-treated beads as well as numbers of hemocytes present at wild-type laser wounds and wounds made to Dp110^D954A-expressing embryos. Error bars represent SEM. (H) Stills from a video showing a bead soaked in LY294002 implanted close to a population of ventral midline hemocytes. The video shows that, although these hemocytes are able to form lamellipodia (arrowhead) and move freely (see Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200508161/DC1), they are unable to chemotax toward the bead, and after 1.5 h, only one hemocyte has managed to detect the bead (arrows). Bars: (A, B, and H) 20 μm; (C–F) 50 μm.