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Fig. 2 Primitive Trim33-deficient macrophages accumulate cytomorphological and metabolic defects before disappearing. (A) Turquoise+ macrophages in the yolk sac of live monTB222/Tg(mfap4:turquoise) control sibling (top) and mutant (bottom) at 3 dpf; maximum projection. (B) Quantification of the roundness of turquoise+ macrophages in live monTB222/Tg(mfap4:turquoise) sibling (left plot, dark grey) and mutant (right plot, light grey) larvae at 3 dpf (n=137 cells from 5 siblings and 79 cells from 3 mutants). ****P<0.0001 Mann–Whitney test. (C) Percentage of turquoise+ macrophages with none, one, two or at least three ramifications in live monTB222/Tg(mfap4:turquoise) sibling and mutant at 3 dpf (n=127 cells from siblings and 86 cells from mutants). Error bars indicate s.e.m. (D) mCherry+ macrophages in the heart region of 3-day-old live monTB222/Tg(mfap4:mCherry-F) control sibling (top) and mutant (bottom); single confocal plane, rostral towards the left. Purple arrowheads indicate macrophages. (E) mCherry+ macrophages in the caudal region of 3-day-old live monTB222/Tg(mfap4:mCherry-F) control sibling (left) and mutant (right), maximum projection, rostral towards the left. Blue asterisk labels a pigment cell. (F,G) Quantification of macrophage fluorescence in live monTB222/Tg(mfap4:mCherry-F) sibling and mutant larvae at 3 dpf, based on (G) fluorescence area (number of fluorescent pixels per macrophage) and (F) total fluorescence (sum of pixel intensities per macrophage); n=10 cells per condition. Data are mean±s.e.m. ****P<0.0001 Mann–Whitney test. (H) Macrophages in the caudal region of 3-day-old live control siblings (top) and monTB222 mutants (bottom) expressing the mfap4:turquoise (macrophages, cyan channel) and mfap4:mCherry-F (macrophage membranes, red channel) transgenes; single confocal plane, rostral towards the left. Purple arrowheads indicate mCherry-F accumulation inside macrophages. (I) Quantification of fluorescence (pixel intensity) along cross-sections of macrophages in mfap4:turquoise/Tg(mfap4:mCherry-F) live sibling (top) and mutant (bottom) at 3 dpf. Black arrowheads indicate membrane-associated mCherry-F signal surrounding the whole-cell turquoise signal in sibling cells. Purple arrowheads indicate mCherry-F intracellular accumulation. Images used for plotting and other examples are shown in Fig. S2. (J) VE-DIC/Nomarski imaging of macrophages in the caudal region of 3-day-old live wild type (top two images) and monNQ039 mutants (bottom four images); rostral towards the left. Purple arrowheads indicate refractile vesicle accumulation inside mutant macrophages. Black arrowheads indicate macrophages in a wild-type sibling. (K) Macrophages in the caudal region of 3-day-old live monTB222 Tg(mfap4:mCherry-F) (macrophages, red channel) stained with LysoID-green (acidic compartments, green channel); control siblings (left) and mutants (right), single plane, lateral views. Purple arrowheads indicate the mCherry-F intracytoplasmic accumulations that also appear to be acidic (LysoID+) and to coincide with the refractile vesicles in mutant macrophages. (L) Bright-field and turquoise fluorescence signal of a dead macrophage in the trunk region of a live monTB222/Tg(mfap4:turquoise) mutant at 72 hpf, single confocal plane, rostral towards the left. (M) Selected time points of an in vivo time-lapse confocal imaging sequence in the caudal region of a monTB222/Tg(mfap4:turquoise) mutant at 72 hpf (rostral towards the left). Yellow arrowheads indicate a macrophage that dies by bursting into apoptotic bodies. y, yolk sac; H, head; h, heart; CA, caudal artery; CHT, caudal hematopoietic tissue; CV, caudal vein; nc, notochord; Nb, number. Scale bars: 50 µm in A,D,E,H,J,K; 10 µm in L.