The zebrafish heart is seeded with macrophages during embryonic stages.

A) Confocal maximum projections of Tg(myl7:GFP); Tg(mpeg1.1:mCherry) (top) and Tg(myl7:mCherry); TgBAC(csf1ra:GFP) (bottom) double transgenic hearts at 5 and 17 dpf. In both cases the myocardium (myl7) is shown in grey. mpeg1.1+ cells are in magenta and csf1ra+ cells in cyan. Colour-coded boxed regions indicate the position of insets. White arrowheads in insets show location of mpeg1.1 and csf1ra-positive macrophages. v = ventricle, a = atrium. Scale bars = 25 μm and 5 μm for insets. B,C) Graphs indicating the number of csf1ra± and mpeg1.1± cells in larval hearts from (B) 2-24 dpf and (C) 2-10 dpf (same data). N = 6-10 larvae per timepoint. Statistical analysis in B: Kruskal-Wallis with Dunn’s multiple comparisons test for each cell population timecourse separately. Statistical analysis in C: Mann-Whitney test between the two cell populations at each timepoint. Ns = not significant. D) Bar charts showing the proportion of mpeg1.1+ (left) and csf1ra+ (right) cells in the ventricle and atrium at the timepoints indicated. The distribution of cells within each chamber is also indicated. Numbers above bars indicate total macrophage number counted. N = 5-12 larvae per timepoint.

Embryonic cTMs are retained at juvenile stages and are primitive haematopoiesis derived.

A) Confocal maximum projection image of a Tg(csf1ra:Gal4i)); Tg(UAS:kaede) larval heart at 21 dpf after photoconversion at 2 dpf. Scale bars = 50 μm Zoomed insets show photoconverted (yellow) csf1ra-positive cells in the i) ventricle and ii) atrium. Scale bars = 10 μm. B) Quantification of csf1ra unphotoconverted (cyan) and photoconverted (yellow) macrophages at 21 dpf, n = 3. C) Maximum projection images of csf1raj4e1/j4e1; TgBAC(csf1ra:GFP) larval hearts at 3 and 5 dpf. An anti-myosin antibody was used to label the myocardium. Scale bars = 25 µm. D) Graph showing quantification of csf1ra macrophage number in hearts of wildtype and csf1raj4e1/j4e1; TgBAC(csf1ra:GFP) hearts at the time-points indicated. N = 8 per genotype. Statistical analysis = Welch’s t-tests between genotype per timepoint. (E,F) Maximum projection images (E) and quantification (F) of the number of L-plastin+ cells in the heart of cmyb−/− larvae and their siblings (het/wt) at 5 dpf. N = 9/8. Statistical analysis = Welch’s t-test.

Subpopulations of MNPs can be identified in the adult zebrafish heart via Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) transgenic lines.

A,B) 3D still views of Imaris renders (taken from supplementary videos 1 & 2, respectively) of individual mpeg1.1+ (magenta) cells in the ventricle associated with myl7+ cardiomyocytes (A) or kdrl+ endothelial cells (B; both shown in grey). Yellow asterisks in A demark individual CMs in direct contact with the mpeg1.1+ macrophage. Scale bar = 10 µm. C) Wholemount imaging of a Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) double transgenic adult heart. The dotted line demarcates the ventricle (V), atrium (A) and bulbus arteriosus (BA). Scale bar = 200 μm. D) High magnification views of individual cells from the ventricle showing differing cell morphology between subtypes. E) Graphical representation of the relative proportions of labelled cells found in the ventricle and atrium. F) Number of cellular projections per cell type. Statistical analysis performed using a Kruskal-Wallis test. Projections counted manually from 28-46 cells per cell type across three ventricles. G) Automated cell shape analysis of mpeg1.1± csf1ra± cells from a 388 µm2 Z-stack imaged within the ventricle. Cells were classified by their expression of mpeg1.1:mCherry and/or csf1ra:GFP and individual cell volume, circularity, and diameter was measured. Statistical analysis was performed by Brown-Forsythe and Welch’s ANOVA test following removal of outliers by the ROUT outlier test (Q = 1%). 50-147 cells measured across three hearts.

Analysis of immune cell distribution in wildtype and csf1raj4e1/j4e1; Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) adult hearts.

A) 3D reconstruction of a cleared adult wildtype heart fluorescently stained with anti-GFP (csf1ra) and anti-mCherry (mpeg1.1) antibodies showing front and side views. Scale bars = 100 µm. B) Single, representative z-slices from cleared Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) ventricles at specified depths (0-400 µm). Insets show enlarged regions of boxed cells. Scale bars (main and inset) = 20 µm. C) Graph indicating mean frequency ± SD of csf1ra:GFP± mpeg1.1:mCherry± populations per 100 µm sub-stack depth through the ventricle of wildtype and csf1raj4e1/j4e1; Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) fish. The Z-stack was divided into 200 x 200 x 100 µm (xyz) sub-stacks and csf1ra:GFP± mpeg1.1:mCherry± cells were manually counted using the Cell Counter plugin on Fiji following blinding of datasets. All cells were counted per field of view, n= 6 per genotype. Statistical analysis was performed on mean frequency per fish by two-way ANOVA with Šidák’s multiple comparisons test. D) Representative images of fluorescent cells on the ventricular surface of wildtype and and csf1raj4e1/j4e1; Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) hearts. E) Automated quantification of csf1ra:GFP+ cell number per 1000 µm2 of the ventricle surface. N = 5 per genotype. Statistical analysis was performed by Kruskal-Wallis test. F) As in (B), single, representative z-slices from csf1raj4e1/j4e1; Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) mutant ventricles at specified depths. Insets show enlarged regions of boxed cells. Scale bar = 20 µm.

csf1ra+ MNPs exhibit differing morphologies and marker expression.

A,B) Flow cytometry plot of cell populations gated by GFP and mCherry expression from Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) hearts (left plot) and corresponding FSC and SSC of these populations (all gates merged in right plot, individual populations shown in B). C) May-Grünwald Giemsa staining of sorted csf1ra± and mpeg1.1± sub-populations. Cells were isolated from 6-8 pooled Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) hearts. Scale bar = 20 µm. D) RT-PCR analysis of sorted populations using MNP related markers. cDNA generated from whole kidney marrow was used as a positive control; water was used for the negative control. Cells were sorted from pools of 15 hearts for each group. Cell numbers: csf1ra+ = 3300; mpeg1.1+csf1radim = 4900; mpeg1.1+ csf1ra+ = 30,000; background cells = 250,000. E) RT-PCR analysis of MNP markers from sorted cells isolated from 1 dpi Tg(mpeg1.1:mCherry); TgBAC(csf1ra:GFP) ventricles. Cell counts: mpeg1.1+; csf1ra+ = 7900; csf1ra+ = 712.

Ex vivo live imaging of cardiac csf1ra± and mpeg1.1± cells reveals different behaviours.

A) Schematic of the ex vivo imaging platform. Single hearts were placed in a custom mould (green) and mounted in 4% low-gelling agarose to limit transverse movement of the heart during imaging. B) Maximum projection of csf1ra± and mpeg1.1± positive cells on the surface of the ventricle. Two mpeg1.1+ csf1ra+ (1/2; grey) cells and two csf1ra+ cells (3/4; cyan/blue) are highlighted. Scale bar = 20 µm. B’) Colour coded tracks of cells 1-4 from B. Cells were tracked from 0 – 100 minutes using the Manual Cell Tracking Plugin on Fiji. C) Maximum projection images of the boxed region in B at the timepoints indicated. Cells 2 (mpeg1.1+, csf1ra+) and 4 (csf1ra+) are indicated in each frame. Scale bar = 20 µm. D) Quantification of mpeg1.1+ csf1ra+ and csf1ra+ MNP behaviour. n = 3 (total cell number analysed: csf1ra+ = 12-51; mpeg1.1+ csf1ra+ = 47-90).

Ventricular mpeg1.1± csf1ra± cells proliferate in response to cardiac injury in wildtype and csf1raj4e1/j4e1fish.

A-C) Frequency of each FAC sorted csf1ra+ subpopulation expressed as a percentage of labelled cells in wildtype and csf1raj4e1/j4e1ventricles at the timepoints indicated. N = 6-8 per timepoint and condition. Statistical analyses = Mann-Whitney pairwise test between experimental groups (blue); Welch’s pairwise test between experimental groups (black). (D,E) Representative single channel and merge channel images of wild type (D) and csf1raj4e1/j4e1hearts (E) at 1 dpi and 7 dpi following labelling with anti-GFP (csf1ra+ - cyan), anti-mCherry (mpeg1.1+ - magenta) and anti-PH3 (proliferating cells – yellow) antibodies. Insets show examples of proliferating MNPs identified by the boxes. Scale bars = 100 µm and 5 µm for insets. F) Quantification of proliferating (PH3+) cells within each genotype at each timepoint. The total number of PH3+ cells were manually counted, grouped by their co-expression with mpeg1.1 and csf1ra+, per 580 μm x 580 μm x 500 μm (xyz) stack acquired at the border zone. N = 5-7 per condition. Statistical analyses = Mann-Whitney pairwise test between experimental groups (blue); Kruskal-Wallis with Dunn’s multiple comparison tests between all timepoints within each experimental group (black). Comparisons without error bars were not significant. G-I) Representative AFOG images of 21 dpi wildtype (G) and csf1raj4e1/j4e1 (H) hearts showing the composition of the injury site. Quantification of injury area (I) shown as the percentage of the ventricle area. For each fish values were averaged from 3 different slides through the injury. N numbers (wildtype/csf1raj4e1/j4e1): 9/11.