Circulating blood cells function as a surveillance system for damaged tissue in Drosophila larvae

Abstract

Insects have an open circulatory system in which the heart pumps blood (hemolymph) into the body cavity, where it directly bathes the internal organs and epidermis. The blood contains free and tissue-bound immune cells that function in the inflammatory response. Here, we use live imaging of transgenic Drosophila larvae with fluorescently labeled blood cells (hemocytes) to investigate the circulatory dynamics of larval blood cells and their response to tissue injury. We find that, under normal conditions, the free cells rapidly circulate, whereas the tissue-bound cells are sessile. After epidermal wounding, tissue-bound cells around the wound site remain sessile and unresponsive, whereas circulating cells are rapidly recruited to the site of damage by adhesive capture. After capture, these cells distribute across the wound, appear phagocytically active, and are subsequently released back into circulation by the healing epidermis. The results demonstrate that circulating cells function as a surveillance system that monitors larval tissues for damage, and that adhesive capture, an important mechanism of recruitment of circulating cells to inflammatory sites in vertebrates, is shared by insects and vertebrates despite the vastly different architectures of their circulatory systems.

Fig. 1.

Circulating and tissue-bound blood cell populations in Drosophila larvae. (A) Schematic of third-instar larva viewed dorsally showing tracheae (black) including dorsal trunks (arrowheads). Blue box, approximate area of view in B–D and Movies S2–S4. Red oval approximate position and size of pinch wounds shown in Figs. 23–4. (B–H) Composite trajectories from movies (B–D), micrographs (E and F) or still frames from movies (G and H) of Nrg-GFP;Pxn>YFP larvae with YFP-labeled blood cells. (B and C) Movement of blood cells in the dorsal (B, from Movie S2) and ventral (C, from Movie S3) sides tracked by videomicroscopy over an ≈8-s interval. Sessile tissue-bound blood cells (red dots) and the trajectories of circulating cells flowing posteriorly through the body cavity (green arrows) are shown. Arrowheads, tracheal dorsal trunks; arrow, ventral nerve cord. (D) Trajectories (yellow arrows) of cells (from Movie S5) pumped anteriorly through the heart and tracked over a 2-s interval. Tissue-bound cells indicated as above. Arrowheads, tracheal dorsal trunks. (E and F) Micrographs of larval anterior (E) and posterior (F) showing pooling of free posterior blood cells. (G) Frames from Movie S1 showing a cluster of three tissue-bound blood cells swivelling about its attachment (arrowhead) to the epidermis. Elapsed time is indicated. (H) Frames from Movie S6 showing transient release into circulation of a small cluster of tissue-bound blood cells that rebinds 4 s later to a tracheal dorsal trunk. Horizontal arrowhead in each frame, tracheal dorsal trunk; red arrowheads, released blood cell cluster. Elapsed time is indicated. [Scale bar (D) 100 μm for B–D; (F) 100 μm for E and F; (G) 50 μm; (H) 100 μm.]

Fig. 2.

Accumulation and turnover of blood cells at larval wounds. (A–D) Unwounded segment (A) or pinch-wounded segments (B–D) in epidermal whole-mount preparations of Pxn>GFP third-instar larvae stained with anti-Fasciclin-III (red) to show epidermal membranes and anti-GFP (green) to show blood cells. (A) Unwounded control larva; (B) ≈5 min after wounding; (C) 4 h after wounding; (D) 24 h after wounding; (E and F) SEM; (E) unwounded epidermis (white dotted ovals, epidermal cell nuclei); (F) exposed cuticle and cell debris ≈5 min after wounding; (G and H) TEM; (G) unwounded epidermis (ep) and apical cuticle (cu); (H) intact cuticle and cell debris (de) ≈5 min after wounding; (I) area of epidermis (+/− SEM) occupied by blood cells in wounded dorsal segments (black bars), adjacent segments located anterior (white bars), and posterior (gray bars). [Scale bar (D) 100 μm for A–D;. (F) 33 μm for F, 26.5 μm for E; (H) 2 μm for G and H.]

Fig. 3.

Direct capture of circulating blood cells at larval wound sites. (A–C) Composite trajectory (A) or still frames (B and C) from movies of wounded Nrg-GFP;Pxn>YFP larvae. Wound sites are outlined in white. Elapsed time is indicated; anterior is up. (A) Track of a circulating blood cell cluster (black line with diamonds) over a 22-s interval from a real-time movie (Movie S10). The cluster moves rapidly through the body cavity until docking abruptly at the wound site. (B) Frames from time-lapse movie (Movie S9). Few blood cells are present at the wound site <5 min after wounding (Upper). A cluster of ≈100 blood cells (white arrowhead) attaches to the wound site over a 2-min interval (32–34 min). Over the next 2 hr, the cluster shears apart, and some cells readhere to downstream (more posterior) portions of the wound (black arrowhead, 152 min). (B′) Closeups of frames from Movie S9 showing a group of sessile tissue-bound cells (arrowhead) near the wound edge (dashed white line) that fail to polarize or migrate to the wound. (C) Frames from time-lapse movie (Movie S11) showing direct docking of a large sheet of blood cells (right half of wound) within a 10-min interval.

Fig. 4.

Morphology of wound-adherent blood cells and release of bound cells by the spreading epidermis. (A–D) SEM (A and B) and TEM (C and D) of blood cells bound at wound sites. (A) Cluster of tissue-bound blood cells in control unwounded larva; (B) blood cells attached to wound site 4 h after wounding; (C) 2 h postwounding. Phagocytic processes (arrowheads) extend from blood cells to engulf cellular debris (asterisk). Arrows, close apposition of blood cells; (C′) closeup of left box in C. Vesciculate cell debris at wound site (upper asterisk) and within a blood cell vesicle surrounded by healthy dark gray cytoplasm (lower asterisk); (C″) closeup of right box in C. Arrowheads, overlapping cell extensions along wound-site debris; (D) 4 h postwounding. Cluster of blood cells under exposed cuticle (cu) of pinch wound. Cell debris (de); (D′) closeup of left box in D. Fine cell processes (arrowheads) attached to debris beneath the cuticle; (D″) closeup of right box in D. Fine cell extensions (arrowheads) between blood cells; (E and F) epidermal whole-mount preparations of pinch-wounded UAS-srcGFP, A58-Gal4 larvae immunostained with anti-GFP (red) to label epidermal cell membranes and anti-Peroxidasin (green) to label blood cells. (E) Control larva lacking UAS-bsk^DN transgene, 24 h after wounding. (F) Larva carrying a UAS-bsk^DN transgene, 24 h after wounding. [Scale bar (B) 10 μm for A and B; (C) 10 μm; (C″), 2 μm for C′, C″; (D) 2 μm; (D″), 1 μm for D′, D″); (F) 100 μm for E and F.