Fig. 1. Yolk-sac-derived and fetal liver-derived macrophage populations are present in the embryonic testis.

a Flow cytometric (FACS) analyses of testicular macrophage populations in embryonic (E) and newborn (NB) wild-type (WT) mice. Shown are representative FACS plots and frequencies of F4/80^Hi (red) and F4/80^Int (blue) testicular macrophage populations at the indicated timepoints. b, c Representative histograms of CD115, CD206, and Ly6C expression in F4/80^Hi and F4/80^Int testicular macrophage populations in b E16.5 and c NB WT mice. d Analysis of F4/80^Hi and F4/80^Int testicular macrophages in E17.5 WT embryo treated with blocking CD115 antibody or control IgG at E6.5. Experimental outline, representative FACS plots, and frequencies of F4/80^Hi and F4/80^Int are shown. e t-SNE maps from mass cytometric analyses displaying the expression of the indicated antigens in randomly sampled live, single CD45⁺CD11b⁺F4/80⁺ cells in testes of NB WT mice. The scale bar indicates the expression level of a given antigen from low (blue) to high (red). f Manual clustering of cell populations based on the expression of known myeloid markers shown in (e). MAC macrophage, MO monocyte. g Unsupervised hierarchical X-shift clustering (nearest neighbor) illustration of macrophage populations from (e). The colored boxes show the location of manually gated cell populations based on the expression analyses of myeloid cell-selective markers (shown in Supplementary Fig. 2i). In the quantifications (a, d), each dot represents a pool of 2–8 testes from 1–4 mice (E14.5, n = 9, and E16.5, n = 8 pools), or both testes of one mouse (E17.5, CO; n = 9, CD115; n = 7 and NB, n = 5 mice). Data are presented as mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, Two-tailed Mann–Whitney U test). All data are from 2–3 independent experiments. Mass cytometry data (e–g) is from 6–10 testes each pooled from 3–5 mice from two independent experiments. Source data are provided as a Source Data file.