Overexposure to apoptosis via disrupted glial specification perturbs Drosophila macrophage function and reveals roles of the CNS during injury

Abstract

Apoptotic cell clearance by phagocytes is a fundamental process during development, homeostasis and the resolution of inflammation. However, the demands placed on phagocytic cells such as macrophages by this process, and the limitations these interactions impose on subsequent cellular behaviours are not yet clear. Here, we seek to understand how apoptotic cells affect macrophage function in the context of a genetically tractable Drosophila model in which macrophages encounter excessive amounts of apoptotic cells. Loss of the glial-specific transcription factor Repo prevents glia from contributing to apoptotic cell clearance in the developing embryo. We show that this leads to the challenge of macrophages with large numbers of apoptotic cells in vivo. As a consequence, macrophages become highly vacuolated with cleared apoptotic cells, and their developmental dispersal and migration is perturbed. We also show that the requirement to deal with excess apoptosis caused by a loss of repo function leads to impaired inflammatory responses to injury. However, in contrast to migratory phenotypes, defects in wound responses cannot be rescued by preventing apoptosis from occurring within a repo mutant background. In investigating the underlying cause of these impaired inflammatory responses, we demonstrate that wound-induced calcium waves propagate into surrounding tissues, including neurons and glia of the ventral nerve cord, which exhibit striking calcium waves on wounding, revealing a previously unanticipated contribution of these cells during responses to injury. Taken together, these results demonstrate important insights into macrophage biology and how repo mutants can be used to study macrophage-apoptotic cell interactions in the fly embryo. Furthermore, this work shows how these multipurpose cells can be 'overtasked' to the detriment of their other functions, alongside providing new insights into which cells govern macrophage responses to injury in vivo.

Fig. 1. Interaction of macrophages and glial cells during Drosophila embryonic development.

a, b Single z-slices (a, b) and maximum projections (a’, b’) of immunostained control embryos with GFP-labelled macrophages (green) showing progression of macrophages along both sides of the ventral nerve cord (VNC, Futch staining; magenta) at stage 12 (a) and 14 (b). Arrows indicate the position of VNC; embryos are laterally orientated with anterior to the left, and ventral down. c Ventral views of a stage 15 control embryo immunostained for apoptotic cells (anti-cDCP-1, magenta) and macrophages (anti-GFP, green); panels show single z-slices showing engulfment of apoptotic cells by macrophages (arrows, c) and apoptotic cells within the VNC (arrowheads, c’), and a maximum projection of this region corresponding to a 20-μm deep z-stack (c”); a white line in (c’) indicates the edges of the VNC—macrophages within these lines are sitting within the midline pores that span this structure. d, e Ventral views of stage 15 control and repo mutant embryos immunostained for GFP to show macrophages (d, e) and anti-Repo (d’, e’); macrophages and Repo are green and magenta, respectively, in merged images (d”, e”); arrows in anti-Repo channel indicate non-nuclear staining likely to be cross-reactivity to another epitope. All scale bars represent 10 μm. Genotypes are as follows: w;srp-GAL4,UAS-GFP/ + ;crq-GAL4,UAS-GFP/ + (a, b), w;;crq-GAL4,UAS-GFP (c, d), w;;repo⁰³⁷⁰²,crq-GAL4,UAS-GFP (e).

Fig. 2. Increased macrophage-mediated apoptotic cell clearance in the absence of glial specification.

a, b Ventral views of stage 15 control (a) and repo mutant (b) embryos containing GFP and red stinger-expressing macrophages; lower panels show zooms of macrophages indicated by boxes in upper panels. c Scattergraph of vacuoles per macrophage per embryo from genotypes in (a, b); P < 0.0001 via Mann–Whitney test (n = 24 and 30). d, e Ventral views of stage 15 control (d) and repo mutant (e) embryos containing GFP-labelled macrophages immunostained for GFP (green in merge) and apoptotic cells (anti-cDCP-1, magenta in merge); zooms show macrophages indicated in boxed regions. f Scattergraphs of phagocytic index (cDCP-1 puncta per macrophage per embryo); P < 0.0001 (n = 101 and 69 cells from 6 and 5 embryos, respectively) and P = 0.0043 (n = 6 and 5 embryos, respectively) via Mann–Whitney tests. Scale bars represent 20 μm or 5 μm in zooms; error bars and lines show standard deviation and mean, respectively (c, f); genotypes are w;srp-GAL4,UAS-GFP/srp-GAL4,UAS-red stinger (a, c), w;srp-GAL4,UAS-GFP/srp-GAL4,UAS-red stinger;repo⁰³⁷⁰² (b, c), w;;crq-GAL4,UAS-GFP (d, f) and w;;repo⁰³⁷⁰²,crq-GAL4,UAS-GFP (e, f).

Fig. 3. Excessive amounts of apoptotic cell death impair macrophage dispersal.

a Lateral views of stage 13/14 control, repo, simu and simu;repo double-mutant embryos containing GFP-labelled macrophages. A complete line of macrophages is present on midline on the ventral side of the VNC in all genotypes, with the exception of simu;repo double mutants (gap indicated via an asterisk); a’ shows zoom of this region. b Scattergraph showing quantification of midline progression defects (numbers of segments lacking macrophages on the ventral side of the VNC); P < 0.0001 (for simu;repo vs each other genotype; no other comparisons are significantly different) via a Kruskall–Wallis test with a Dunn’s multiple comparaison post-test; n = 15 (controls), 17 (repo mutants), 15 (simu mutants) and 11 (simu;repo). c, d Ventral views of stage 13 control and repo mutant embryos containing GFP-expressing macrophages (immunostained via anti-GFP). e Scattergraph showing quantification of numbers of macrophages in five central segments on the ventral midline; P < 0.0001 via Mann–Whitney test, n = 13 (controls), 13 (repo mutants). Scale bars represent 50 μm (a) and 25 μm (c, d); lines and error bars on scattergraphs show mean and standard deviation, respectively; **** indicates P < 0.0001. Genotypes are w¹¹¹⁸;;crq-GAL4,UAS-GFP (control), w¹¹¹⁸;;P{PZ}repo⁰³⁷⁰²,crq-GAL4,UAS-GFP (repo⁰³⁷⁰²), w¹¹¹⁸;simu²;crq-GAL4,UAS-GFP (simu²) and w¹¹¹⁸;simu²;P{PZ}repo⁰³⁷⁰²,crq-GAL4,UAS-GFP (simu;repo).

Fig. 4. Excessive amounts of apoptotic cells impair macrophage migration.

a–d Ventral images of GFP and red stinger-labelled macrophages (green and magenta, respectively) in stage 15 control (a), Df(3L)H99 mutant (b), repo⁰³⁷⁰² mutant (c) and Df(3L)H99,repo⁰³⁷⁰² double-mutant embryos (d). Upper panels show initial position of macrophages on the ventral midline; central panels show tracks taken from subsequent 60-min period from the timepoint shown in the upper panel; the lower panel shows overlay of tracks over initial positions of macrophages. e Superplot showing speed per macrophage per embryo (black dots) in μm per min; grey dots represent individual cell speeds to show range of migration speeds in each genotype. Lines and error bars show mean and standard deviation, respectively (embryo averages). Removing apoptosis from a repo mutant background (Df(3L)H99,repo) rescues migration speed (P = 0.0095 via Mann–Whitney test; n = 4, 5, 6 and 6 embryos (left to right)). f, g Scatterplots of macrophage speed and vacuolation for macrophages in control (f) and repo mutant (g) embryos (80 and 103 macrophages analysed from 6 and 10 control and repo mutant embryos, respectively). Linear regression lines shown in magenta. Scale bars represent 20 μm; ns and ** indicate not significant and P < 0.01, respectively. Genotypes are w¹¹¹⁸;srp-GAL4,UAS-red stinger/srp-GAL4,UAS-GFP (control), w¹¹¹⁸;srp-GAL4,UAS-red stinger/srp-GAL4,UAS-GFP;Df(3L)H99 (Df(3L)H99), w¹¹¹⁸;srp-GAL4,UAS-red stinger/srp-GAL4,UAS-GFP;P{PZ}repo⁰³⁷⁰² (repo⁰³⁷⁰²) and w¹¹¹⁸;srp-GAL4,UAS-red stinger/srp-GAL4,UAS-GFP;Df(3L)H99,P{PZ}repo⁰³⁷⁰² (Df(3L)H99,repo⁰³⁷⁰²).

Fig. 5. repo is required for normal macrophage inflammatory responses to wounding.

a, b Ventral views showing localisation of GFP-labelled macrophages on the ventral midline in control (a, a’) and repo mutant (b, b’) embryos immediately before wounding (a, b) and at 60-min post wounding (a’, b’); white-dotted ellipses indicate wound edges. c Scattergraph showing numbers of macrophages in the field of view ahead of wounding per embryo; P < 0.0001 via Mann–Whitney test (n = 23 and 15 control and repo, respectively). d Scattergraph showing wound responses quantified via density of macrophages at wounds, normalised to control; P < 0.0001 via Mann–Whitney test (n = 23 and 17 control and repo, respectively). e Scattergraph showing percentage of macrophages responding to wounds; P = 0.0003 via Mann–Whitney test (n = 7 and 9 control and repo, respectively). f Scattergraph showing percentage of macrophages that leave the wound (having initially migrated to the wound); P > 0.999 via Mann–Whitney test (n = 7 and 9 control and repo, respectively). Lines and error bars in scattergraphs show mean and standard deviation, respectively; scale bars represent 20 μm; ns, *** and **** indicate not significant (P > 0.05), P < 0.001 and P < 0.0001, respectively. Genotypes are w;;crq-GAL4,UAS-GFP (control) and w;crq-GAL4,UAS-GFP,repo⁰³⁷⁰² (repo⁰³⁷⁰²).

Fig. 6. Loss of repo impairs wound responses, but contact with apoptotic cells is not the overriding cause of this defect.

a–d Ventral views showing localisation of GFP and red stinger-labelled macrophages on the ventral midline in control (a, a’), Df(3L)H99 (b, b’), repo (c, c’) and Df(3L)H99,repo double-mutant embryos (d, d’) immediately before wounding (a–d) and at 60 min post wounding (a’–d’); white-dotted ellipses indicate wound edges. e Scattergraph showing wound responses quantified via density of macrophages at wounds, normalised to control; Mann–Whitney tests used to compare control vs Df(3L)H99 (n = 15 and 7, respectively; P = 0.63) and repo vs Df(3 L)H99,repo (n = 9 and 15, respectively; P > 0.99). f Scattergraph showing percentage of macrophages responding to wounds; Mann–Whitney tests used to compare control vs Df(3L)H99 (n = 9 and 4, respectively P = 0.055) and repo vs Df(3L)H99,repo (n = 6 and 6, respectively; P = 0.17). Lines and error bars in scattergraphs show mean and standard deviation, respectively; scale bars represent 20 μm; ns indicates not significant (P > 0.05). Genotypes are w;srp-GAL4,UAS-GFP/srp-GAL4,UAS-red stinger (control), w;srp-GAL4,UAS-GFP/srp-GAL4,UAS-red stinger;Df(3L)H99 (Df(3L)H99), w;srp-GAL4,UAS-GFP/srp-GAL4,UAS-red stinger;repo⁰³⁷⁰² (repo⁰³⁷⁰²) and w;srp-GAL4,UAS-GFP/srp-GAL4,UAS-red stinger;Df(3L)H99,repo⁰³⁷⁰² (Df(3L)H99,repo⁰³⁷⁰²).

Fig. 7. Defective calcium responses to wounding in repo mutants are restricted to less superficial tissues.

a, b Calcium levels imaged via ubiquitous expression of GCaMP6M (da-GAL4,UAS-GCaMP6M) on wounding of the ventral surface of control (a) and repo mutant (b) stage 15 embryos; images show pre-wound calcium levels, immediately after wounding (0 min) and 5 min after wounding. Dotted lines show edges of embryo and wound edges in pre-wound and 5 min images, respectively. c Scattergraph showing area of GCaMP6M response immediately after wounding; Mann–Whitney test used to compare control vs repo (n = 35 and 47, respectively; P = 0.66). d Scattergraph showing ratio of GCaMP6M intensity in wound area before and immediately after wounding; Mann–Whitney test used to compare control vs repo (n = 23 and 26, respectively; P = 0.22). e–g maximum projections of superficial/epithelial regions (0–5 μm, e), superficial half of the VNC (10–20 μm from surface, f) and deeper/dorsal half of the VNC (20–30 μm from the surface, g) of the ventral side of a wounded control embryo containing ubiquitous expression of GCaMP6M; panels show pre-wound calcium levels, immediately after wounding (0 min) and 5 min after wounding. Dotted lines show edges of epithelial wound at 5 min, solid circles show physical damage at deeper regions of the embryo; embryo has not been orientated in order to show larger region of the response to wounding. Lines and error bars in scattergraphs show mean and standard deviation, respectively; scale bars represent 20 μm; ns indicates not significant (P > 0.05). Genotypes are w;;da-GAL4,UAS-GCaMP6M (control) and w;;repo⁰³⁷⁰²,da-GAL4,UAS-GCaMP6M (repo).

Fig. 8. Non-epithelial tissues contribute to wound responses.

a, b Calcium levels within the VNC imaged via expression of UAS-GCaMP6M via e22c-GAL4 (projections assembled from slice range 30–10-μm deep from surface of the embryo) on wounding of the ventral surface of control (a) and repo mutant (b) stage 15 embryos; images show pre-wound calcium levels and immediately after wounding (0 min). c Scattergraph showing GCaMP6M response within VNC immediately after wounding (F1/F0) of control and repo mutants labelled using e22c-GAL4,UAS-GCaMP6M; Mann–Whitney test used to compare control vs repo (n = 37 and 30, respectively; P = 0.0051). d Calcium levels in neuronal cells (labelled using elav-GAL4 to drive UAS-GCaMP6M expression) on wounding of the ventral surface of a stage 15 embryo; images show pre-wound calcium levels, immediately after wounding (0 min) and 5-min after wounding. e–g Calcium levels in glial cells (labelled using repo-GAL4 to drive UAS-GCaMP6M expression) on wounding of the ventral surface of control (e, g) and repo (f) mutant stage 15 embryos; images show pre-wound calcium levels, immediately after wounding (0 min) and 5-min after wounding. g shows embryo wounded more laterally and subsequent spread of calcium signal along glial cells to more lateral positions; dotted line shows equivalent position in the 0 min and 2 min post-wound timepoint images. h, i Scattergraphs showing GCaMP6M responses in glial cells immediately after wounding (h, F1/F0) and the initial area (μm²) of the GCaMP6M response in control and repo mutant embryos labelled via repo-GAL4,UAS-GCaMP6M (i); Mann–Whitney test used to compare control vs repo (n = 16 and 12, respectively; P = 0.42 (h) and P = 0.0003 (i)). Asterisks show position of wounds in pre-wound images; scale bars represent 20 μm; lines and error bars show mean and standard deviation in all scattergraphs; **, *** and ns denote P < 0.01, P < 0.001 and not significant (P > 0.05), respectively. Genotypes are w;e22c-GAL4,UAS-GCaMP6M (a, c), w;e22c-GAL4,UAS-GCaMP6M;repo⁰³⁷⁰² (b, c), elav-GAL4/w;UAS-GCaMP6M/+ (d), w;repo-GAL4,UAS-GCaMP6M (e, g, h–i) and w;repo-GAL4,UAS-GCaMP6M;repo⁰³⁷⁰² (f, h, i).