Extended Data Fig. 9: Tissue-resident macrophages in the developing testes.

a, Schematics illustrating the CD45+ enrichment strategy for gonadal and extragonadal samples. The 11 samples that were sorted with the pan-leukocyte marker CD45 are from the following developmental stages: 6, 11, 12 PCW males, and 7.5, 8.4, late 8, 9, 11, 11, 14, 17 PCW females. b, Gating strategy to sort immune cells in gonadal samples for a representative donor (F93). Cells were gated on live, singlets and CD45+. c, UMAP projections of immune cells labelled by sex, PCW and donor. d, Heatmap showing label transfer scores from the fetal liver hematopoiesis dataset (Popescu et al., 2019) to our gonadal immune dataset using a Support Vector Machine (SVM) classifier. Low probabilities assigned to neutrophils, which were not defined in the liver dataset, and to macrophages e, Dot plot showing variance-scaled, log-transformed expression of marker genes expressed in the identified immune subsets. f, Barplot showcasing the proportions of immune cells labelled by cell state and classified by sex and developmental stage. g, Barplot showcasing the proportion of cells belonging to each identified macrophage population in females and males. h, Dot plot showing the variance-scaled, log-transformed expression of microglia markers in the cluster of TREM2+ ftM in both sexes reveals that the few female cells that belong to this cluster do not express the key markers. i, Predicted probability of bone marrow osteoclasts from Jardine et al., 2021 (left) and brain microglia cells from Bian et al., 2020 (right) onto our gonadal immune manifold using a SVM classifier. j, UMAP (uniform manifold approximation and projection) of the multi-organ integrated fetal myeloid dataset labelled by tissue and donor. k, Dot plot showing variance-scaled, log-transformed expression of marker genes expressed in the identified cell populations from the multi-organ integrated fetal myeloid dataset. l, Dot plots showing variance-scaled, log-transformed expression of interacting ligands and receptors in the SIGLEC15+ and TREM2+ ftM and gonadal cell populations. Interacting partners (CellPhoneDB) are indicated with a matching symbol. m, Dot plot showing variance-scaled, log-transformed expression of immunoregulatory markers in human gonadal macrophages. cDC = conventional Dendritic cells; ECM = extracellular matrix; ESGC = early supporting gonadal cells; ftM = fetal testicular macrophages; Gi = gonadal interstitial; ILC prec = innate lymphoid cell precursors; Mac = macrophages; Mast = mast cells; Mega = megakaryocytes; MEMP = megakaryocyte-erythroid-mast cell progenitors; Mono = monocytes; Neutro = neutrophil; NMP = neutrophil-myeloid progenitors; NK = Natural Killer cells; pDC = plasmacytoid Dendritic cell; PV = perivascular; PCW = post-conceptional week; Prec = precursor; Pre_B = pre-B cells; Pre_pro_B = pre-pro-B cells; Pro_B = pro-B cells; Prob_ = probability; sPAX8 = supporting PAX8; Gi = gonadal interstitial; T = T cells; Ti = testicular interstitial.