Apoptotic cell death contributes to developmental dispersal of <i>Drosophila</i> embryonic macrophages.

(a–b) Lateral projections (anterior is left) of control (w;srp-GAL4,UAS-GFP) and apoptosis-null Df(3L)H99 mutant embryos (w;srp-GAL4,UAS-GFP;Df(3L)H99) showing macrophage distribution at stage 13/14 of development, including along ventral midline (arrow). (c–d) Ventral projections (anterior is up) showing macrophages (anti-GFP staining, green in merge) and structure of the CNS (22c10/anti-Futch staining, purple in merge) at stage 13 of development in controls (w;;crq-GAL4,UAS-GFP) and in the absence of apoptosis (w;;Df(3L)H99,crq-GAL4,UAS-GFP). Macrophage projections constructed from z-slices corresponding to superficial macrophages on the ventral side of the VNC only, whereas Futch projection covers the entire volume of the VNC. (e–f) Ventral projections (anterior is up) showing macrophage distribution (GFP) alone (e–f), or merged with Futch staining (e’–f’) to show CNS structure in control and Df(3L)H99 mutant embryos at stage 15 (genotypes as per c–d). (g) Scatterplot showing percentage of macrophages on the midline that move laterally at stage 13 of development in control and apoptosis-null embryos (n = 9 and 10, respectively; P = 0.0006, Mann–Whitney test); lateral migration concludes around the end of stage 14. (h) Scatterplot showing speed per macrophage, per embryo (μm per min) in control and apoptosis-null embryos at stage 13 (P n = 18, 8, 14, 6 [left–right]; P = 0.47, one-way ANOVA with Tukey’s multiple comparison test). Genotypes in (g–h) are as per (c–f). (i) Stills from movie of macrophages phagocytosing at stage 13 on the ventral midline in a control embryo (w;srp-GAL4,UAS-2xFYVE-GFP;crq-GAL4,UAS-CD4-tdTomato). GFP and tdTomato are shown in green and purple, respectively, while asterisk shows nascent phagosome at indicated times (mins:secs) as it becomes positive for the PI3P sensor 2xFYVE-GFP post engulfment; CD4-tdTomato labels membranes. (j–m) Ventral projections of control (w;srp-GAL4,UAS-2xFYVE-GFP;crq-GAL4,UAS-tdTomato) and apoptosis-null embryos (w;srp-GAL4,UAS-2xFYVE-GFP;crq-GAL4,UAS-tdTomato;Df(3L)H99) at stage 13 (j–k) and stage 15 (l–m), showing recent phagocytic events via 2xFYVE-GFP sensor for PI3P. Asterisks show examples of 2xFYVE-GFP positive cells (j, l). (n) Scatterplot showing number of phagocytic events (via 2xFYVE-GFP positive vacuoles) per macrophage, per embryo at stages 13 and 15 (n = 19 and 15, respectively; P P P S1 Data. CNS, central nervous system; GFP, green fluorescent protein; PI3P, phosphatidylinositol 3-phosphate; UAS, upstream activating sequence.