In the testis, interstitial macrophages are thought to be derived from the yolk sac during fetal development, and later replaced by bone marrow-derived macrophages. By contrast, the peritubular macrophages have been reported to emerge first in the postnatal testis and solely represent descendants of bone marrow-derived monocytes. Here, we define new monocyte and macrophage types in the fetal and postnatal testis using high-dimensional single-cell analyses. Our results show that interstitial macrophages have a dominant contribution from fetal liver-derived precursors, while peritubular macrophages are generated already at birth from embryonic precursors. We find that bone marrow-derived monocytes do not substantially contribute to the replenishment of the testicular macrophage pool even after systemic macrophage depletion. The presence of macrophages prenatally, but not postnatally, is necessary for normal spermatogenesis. Our multifaceted data thus challenge the current paradigms in testicular macrophage biology by delineating their differentiation, homeostasis and functions.

Macrophages are required for normal embryogenesis, tissue homeostasis and immunity against microorganisms and tumours. Adult tissue-resident macrophages largely originate from long-lived, self-renewing embryonic precursors and not from haematopoietic stem-cell activity in the bone marrow. Although fate-mapping studies have uncovered a great amount of detail on the origin and kinetics of fetal macrophage development in the yolk sac and liver, the molecules that govern the tissue-specific migration of these cells remain completely unknown. Here we show that an endothelium-specific molecule, plasmalemma vesicle-associated protein (PLVAP), regulates the seeding of fetal monocyte-derived macrophages to tissues in mice. We found that PLVAP-deficient mice have completely normal levels of both yolk-sac- and bone-marrow-derived macrophages, but that fetal liver monocyte-derived macrophage populations were practically missing from tissues. Adult PLVAP-deficient mice show major alterations in macrophage-dependent iron recycling and mammary branching morphogenesis. PLVAP forms diaphragms in the fenestrae of liver sinusoidal endothelium during embryogenesis, interacts with chemoattractants and adhesion molecules and regulates the egress of fetal liver monocytes to the systemic vasculature. Thus, PLVAP selectively controls the exit of macrophage precursors from the fetal liver and, to our knowledge, is the first molecule identified in any organ as regulating the migratory events during embryonic macrophage ontogeny.

Macrophages serve multiple functions including immune regulation, morphogenesis, tissue homeostasis and healing reactions. The current paradigm holds that mammary gland macrophages first arise postnatally during the prepubertal period from the bone marrow-derived monocytes. Here we delineate the origins of tissue-resident mammary gland macrophages using high-dimension phenotypic analyses, cell-fate mapping experiments, gene-deficient mice lacking selective macrophage subtypes, and antibody-based depletion strategies. We show that tissue-resident macrophages are found in mammary glands already before birth, and that the yolk sac-derived and fetal liver-derived macrophages outnumber the adult-derived macrophages in the mammary gland also in the adulthood. In addition, fetal-derived mammary gland macrophages have a characteristic phenotype, display preferential periductal and perivascular localization, and are highly active in scavenging. These findings identify fetal-derived macrophages as the predominant leukocyte type in the adult mammary gland stroma, and reveal previously unknown complexity of macrophage biology in the breast.