Fig 3. Loss of simu function leads to defects in developmental dispersal of macrophages and an early build up of apoptotic cells.

(a–b) Lateral images of control (w;srp-GAL4,UAS-red stinger) and simu mutant embryos (w;simu²,srp-GAL4,UAS-red stinger) showing migration of red stinger-labelled macrophages along the ventral midline at stage 13; arrows indicate ventral midline, anterior is left, ventral is up. (c) Bar graph showing percentage of segments with GFP-labelled macrophages on the ventral side of the VNC at stage 13 in controls (w;;crq-GAL4,UAS-GFP) and simu mutants (w;simu²;crq-GAL4,UAS-GFP) (n = 15 and 15, respectively; P > 0.999, Mann–Whitney test). (d) Total numbers of red stinger-labelled macrophages in lateral views of control and simu mutants (genotypes as per [a–b] at stage 13; n = 7 for each; P = 0.066, Mann–Whitney test). (e–f) GFP-labelled macrophages on the ventral side of the embryo in controls (e) and simu mutants (f); imaging started at the end of stage 12 with time indicating duration of imaging. (g) Numbers of GFP-labelled macrophages on the ventral surface of embryos at the indicated timepoints in control and simu mutants (n = 17 and16, respectively, P = 0.0162 and 0.046 and at 0 and 90 mins, respectively, Student t tests); genotypes as per (c) in e–g. (h–i) Ventral views showing migration of mCherry-labelled macrophages (purple in merge) down the ventral midline (anterior is up) encountering GC3ai-positive apoptotic cells (green in merge) during stage 12 in control and simu mutant embryos. (j) GC3ai fluorescence levels (arbitrary units) quantified from average projections of the ventral midline region of control and simu mutant embryos at stage 12; genotypes are w;;da-GAL4,UAS-GC3ai/srp-3x-mCherry (control) and w;simu²;da-GAL4,UAS-GC3ai/srp-3x-mCherry (simu) in (h–j) (n = 7 and 6, respectively; P = 0.0004, Student t test). Lines/bars and error bars show mean and standard deviation; ns, * and *** denote not significant, P < 0.05 and P < 0.001, respectively; scale bars represent 50 μm (a–b) and 20 μm (e–f, h–i). All data used to plot graphs may be found in Supporting information file S1 Data. GFP, green fluorescent protein; UAS, upstream activating sequence.