Dear Editor

Apoptosis regulation in Drosophila has many parallels to vertebrate systems. However, one major difference has been the apparent lack of mitochondrial significance in Drosophila cell death. Several reports have suggested that mitochondrial outer membrane permeabilization does not occur in Drosophila apoptosis and that there is no significant mitochondrial contribution to apoptosis in the fly.1, 2, 3, 4, 5, 6 However, these findings are based largely, if not exclusively, on studies in cultured S2 cells, which are phagocytic cells derived from embryonic macrophages/hemocytes.7, 8 The specialized nature of S2 cells prompted us to consider whether they are generally representative of Drosophila cells in their response to apoptotic stimuli. We addressed this question by examining the response of embryonic macrophages to apoptotic signals in situ in the embryo.

In keeping with previous accounts, we find that apoptotic S2 cells maintain mitochondrial membrane integrity as measured by CMXRos staining.4 CMXRos is a mitochondrial-specific label dependent on membrane polarization for uptake. Untreated cells show punctate cytoplasmic staining consistent with mitochondrial labeling (Figure 1a). The specificity of labeling is demonstrated by treatment with carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), a protonophore that depolarizes mitochondria by abolishing the proton gradient across the inner mitochondrial membrane.9, 10 S2 cells continue to stain with CMXRos qat high levels even after exposure to UV irradiation and a recovery period of 6 h (Figure 1a). However, this is not the case with Drosophila cells in general. Embryos subjected to ectopic hid expression (Figure 1c–e) or UV irradiation (not shown) rapidly lose the capacity to take up CMXRos in nearly all cells. Within 1 h of the initiation of either signal, nearly all cells of the embryo fail to stain with CMXRos. The notable exception is macrophages, which continue to stain at high levels whereas other cell types do not (Figure 1c–d). These cells are identifiable as macrophages by their stereotypic distribution and enormous size, and by doubly labeling for Croquemort (Figure 1e), a macrophage-specific cell surface marker.11, 12 Identical results were obtained using the mitochondrial marker TMRE (not shown).

Figure 1
figure 1

Embryonic macrophages selectively maintain mitochondrial membrane polarization following induction of apoptosis. (a) S2 cells stained with CMXRos: untreated, mock treated, FCCP treated, UV-irradiated. (b, c) Stage 13 embryos stained with CMXRos and imaged live through the vitelline membrane. (b) CyO/CyO, (c) hs-hid/CyO. Embryos in (b and c) were subjected to 60 min of heat shock before staining. Embryos in (b and c) are siblings and were prepared and imaged together. Genotypes were determined by the intensity of green fluorescent protein (GFP) from a kruppel-GFP CyO balancer. (d) Head region of embryo shown in (c) showing CMXRos-positive macrophages in a field of CMXRos-negative cells. (e) hs-hid/CyO embryo subjected to 60 min of heat shock and labeled with CMXRos and anti-Crq to confirm the identity of CMXRos-positive cells. Image is of the extreme anterior portion of the embryonic head. (f) TEM of 90 nm thin section through the head region of a stage 13 hs-hid embryo subjected to 60 min of heat shock before fixation. Low-magnification image of region showing a healthy macrophage (large cell, boxed) surrounded by apoptotic corpses (small cell boxed; asterisks). Higher magnification images of boxed cells are shown in Supplementary Figure 1

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These findings suggest that macrophages have the capacity to remain healthy in the presence of apoptotic signals that readily affect other cell types. We addressed this possibility by examining the ultrastructure of macrophages in apoptotic embryos by transmission electron microscopy. We found that after 1 h of heat shock-induced hid expression, macrophages in the embryonic head showed no overt signs of apoptosis, while surrounded by apoptotic corpses derived from other cell types (Figure 1f; Supplementary Figure 1). Macrophage chromatin remained uncondensed with the nuclear envelope intact, and membrane-bound organelles including mitochondria, Golgi and endoplasmic reticulum were clearly visible in the cytoplasm. By contrast, surrounding corpses showed condensed chromatin, degeneration of the nuclear envelope and loss of subcellular organelles (Figure 1f; Supplementary Figure 1). These results are consistent with observations by us and others that following exposure to UV irradiation, Croquemort-positive cells in the embryo become quite large owing to multiple engulfment events (R Hays, unpublished finding).11 One would expect macrophages to undergo apoptosis like any other cell type, having been exposed to the same dose of radiation. However, they survive to engulf corpses for a period of at least several hours.

These findings suggest that macrophages are uniquely resistant to apoptosis among embryonic cell types, and that S2 cells, an embryonic macrophage line, is not a suitable general model for Drosophila cell death. They further suggest that apoptosis regulation in Drosophila has more in common with vertebrate systems than previously appreciated, and re-shape a large segment of current Drosophila apoptosis models.