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. 2021 Apr 22;10:e58686. doi: 10.7554/eLife.58686

Figure 8. Drosophila plasmatocyte subpopulations demonstrate functional differences compared to the overall plasmatocyte population.

(a–b) Example images showing plasmatocyte wound responses at 60 min post-wounding (maximum projections of 15 μm deep regions). Cells labelled via UAS-stinger using srpHemo-GAL4 (a) and VT17559-GAL4 (b); dotted lines show wound edges. (c–d) Scatterplots showing percentage of srpHemo-GAL4 (control) or VT-GAL4-labelled plasmatocytes responding to wounds at 60 min (c) or total numbers of labelled plasmatocytes in wounded region (d); p=0.018, 0.041, 0.99, 0.0075 compared to srpHemo-GAL4 (n = 77, 21, 22, 26, 25) (c); p<0.0001 compared to srpHemo-GAL4 for all lines (n = 139, 35, 37, 30, 44) (d). (e–f) Example tracks of plasmatocytes labelled with GFP via srpHemo-GAL4 (e) and VT17559-GAL4 (f) during random migration on the ventral side of the embryo for 1 hr at stage 15. (g–h) Scatterplots showing speed per plasmatocyte, per embryo (g) and directionality (h) at stage 15 in embryos containing cells labelled via srpHemo-GAL4 (control) or the VT-GAL4 lines indicated; p=0.0097, 0.999, 0.82, 0.226 compared to srpHemo-GAL4 (n = 21, 19, 17, 21, 20) (g); p=0.998, 0.216, 0.480, 0.999 compared to srpHemo-GAL4 (n = 21, 19, 17, 21, 20) (h). (i–j) Example images of cells on the ventral midline at stage 15 with labelling via UAS-stinger expression using srpHemo-GAL4 (i) and VT17559-GAL4 (j); plasmatocytes shown in close-up images (i’, j’) are indicated by white boxes in main panels; arrows show phagosomal vesicles, ‘n’ marks nucleus; n.b. panels contrast enhanced independently to show plasmatocyte morphology. (k) Scatterplot showing phagosomal vesicles per plasmatocyte, per embryo at stage 15 (measure of efferocytosis/apoptotic cell clearance); cells labelled via srpHemo-GAL4 (control) or the VT-GAL4 lines indicated; p=0.0020, 0.99, 0.0040, 0.0002 compared to srpHemo-GAL4 (n = 76, 10, 12, 29, 31). (l) Scatterplot showing number of times 2x-FYVE-EGFP sensor recruited to phagosomes (FYVE events) per plasmatocyte, per embryo in plasmatocytes labelled via the split GAL4 system; p=0.019, 0.0034, 0.039 and 0.015 compared to srp control (n = 4, 6, 8, 5 and 12 embryos). Lines and error bars represent mean and standard deviation, respectively (all scatterplots); one-way ANOVA with a Dunnett’s multiple comparison test used to compare VT lines with srp controls in all datasets; ns, *, **, and **** denote not significant (p>0.05), p<0.05, p<0.01, and p<0.0001, respectively. All scale bars represent 20 μm. See Supplementary file 1 for full list of genotypes. N.b. Figure 8—figure supplements 1–3 show analysis of subpopulation cell morphology, ROS levels and phagocytosis in response to immune challenge, respectively.

Figure 8—source data 1. Numerical data used to plot panel (c) of Figure 8.
Figure 8—source data 2. Numerical data used to plot panel (d) of Figure 8.
Figure 8—source data 3. Numerical data used to plot panel (g) of Figure 8.
Figure 8—source data 4. Numerical data used to plot panel (h) of Figure 8.
Figure 8—source data 5. Numerical data used to plot panel (k) of Figure 8.
Figure 8—source data 6. Numerical data used to plot panel (l) of Figure 8.

Figure 8.

Figure 8—figure supplement 1. VT-GAL4-labelled subpopulations show no gross differences in morphology compared to non-labelled plasmatocytes.

Figure 8—figure supplement 1.

(a–e) Representative images of plasmatocytes at stage 15 on the ventral midline labelled using the pan-hemocyte marker srpHemo-GMA (green) and UAS-tdTomato (magenta) via srpHemo-GAL4 (a), VT17559-GAL4 (b), VT32897-GAL4 (c), VT57089-GAL4 (d), and VT62766-GAL4 (e); scale bars represent 20 μm. Scatterplots showing plasmatocyte spread area (f), circularity (g), aspect ratio (h), and roundness (i) for positive control cells (labelled via srpHemo-GAL4), and VT-GAL4-positive/negative cells within embryos corresponding to those shown in (a–e). Cells positive for VT-GAL4-driven tdTomato expression were compared to non-tdTomato expressing cells via Student’s t-test. No significant difference (ns) was found for any VT-GAL4 line tested; lines and error bars represent mean and standard deviation, respectively; data points represent individual plasmatocytes taken from a minimum of three embryos. See Supplementary file 1 for full list of genotypes.
Figure 8—figure supplement 1—source data 1. Numerical data used to plot panel (f) of Figure 8—figure supplement 1.
Figure 8—figure supplement 1—source data 2. Numerical data used to plot panel (g) of Figure 8—figure supplement 1.
Figure 8—figure supplement 1—source data 3. Numerical data used to plot panel (h) of Figure 8—figure supplement 1.
Figure 8—figure supplement 1—source data 4. Numerical data used to plot panel (i) of Figure 8—figure supplement 1.
Figure 8—figure supplement 2. VT-GAL4-labelled plasmatocytes show no gross differences in their ROS levels compared to the overall population.

Figure 8—figure supplement 2.

(a–e) Representative images (single z-slices) of VT-GAL4-positive plasmatocytes (labelled via expression from UAS-tdTomato, magenta in merge) at stage 15 on the ventral midline stained via dihydrorhodamine 123 (DHR123) to show ROS levels (green in merge); srpHemo-GAL4 was used as a positive control to show the overall population. (f) Scatterplot showing quantification of mean gray value per srpHemo-GAL4 or VT-GAL4-labelled plasmatocyte, per embryo; lines and error bars represent mean and standard deviation, respectively. No statistically significant differences (p>0.05) were found between the VT-GAL4 lines shown and the overall population (labelled via srpHemo-GAL4) using a one-way ANOVA compared to control; n = 12 (control); n = 11, p>0.99 (VT17559-GAL4); n = 11, p=0.68 (VT32897-GAL4); n = 14, p>0.99 (VT57089-GAL4); n = 14, p=0.53 (VT62766-GAL4) embryos; ns denotes not significantly different to control; scale bars represent 20 μm (a–e). See Supplementary file 1 for full list of genotypes.
Figure 8—figure supplement 2—source data 1. Numerical data used to plot panel (f) of Figure 8—figure supplement 2.
Figure 8—figure supplement 3. VT-GAL4-labelled plasmatocytes show no gross differences in their phagocytosis of E. coli compared to the overall population.

Figure 8—figure supplement 3.

(a) Ventral views of stage 15 embryos containing srpHemo-GAL4 (positive control, overall population) and VT-GAL4-positive plasmatocytes (labelled via expression from UAS-tdTomato, magenta) 1 hr following injection with pHrodo-labelled E. coli particles (green); srpHemo-GAL4 was used as a positive control to show the overall population; srpHemo-GMA used to label VT-GAL4 negative plasmatocytes. (b) Shows close-ups of E. coli-positive plasmatocytes indicated by white boxes in (a). (c) Scatterplot showing quantification of the proportion of pHrodo E. coli-positive plasmatocytes per embryo in the populations labelled via srpHemo-GAL4 or the indicated VT-GAL4 reporter. Lines and error bars represent mean and standard deviation, respectively. No statistically significant difference (ns; p>0.05) was found between the VT-GAL4 lines shown and plasmatocytes labelled via srpHemo-GAL4 using a Kruskal-Wallis test with Dunn’s multiple comparisons test: n = 22 (srpHemo-GAL4); n = 31, p=0.14 (VT17559-GAL4); n = 28, p>0.99 (VT32897-GAL4); n = 29, p>0.99 (VT57089-GAL4); n = 28, p>0.99 (VT62766-GAL4). Scale bars represent 20 μm. See Supplementary file 1 for full list of genotypes.
Figure 8—figure supplement 3—source data 1. Numerical data used to plot panel (c) of Figure 8—figure supplement 3.