(and to right of is a higher-magnification image of the cell denoted by the open arrowhead in indicating internalized vascular material. macrophages in fetal organogenesis that may be relevant to the development of many organs. in pre-Sertoli cells directs the gonad toward a male-specific fate. The cells in the early bipotential gonad undergo de novo organization to form testis cords that enclose germ cells inside tubules lined by epithelial Sertoli cells. Although Sertoli cells are a driving force in the de novo formation of testis cords, recent studies in mouse showed that reorganization of the vasculature and of interstitial cells also play critical roles CL-387785 (EKI-785) in testis cord morphogenesis. However, the mechanism driving reorganization of the vasculature during fetal organogenesis remained unclear. Here we demonstrate that fetal macrophages are associated with nascent gonadal and mesonephric vasculature during the initial phases of testis morphogenesis. Macrophages mediate vascular reorganization and prune errant germ cells and somatic cells after testis architecture is established. We show that gonadal macrophages are derived from primitive yolk-sac hematopoietic progenitors and exhibit hallmarks of M2 activation status, suggestive of angiogenic and tissue remodeling functions. Depletion of macrophages resulted in impaired vascular reorganization and abnormal cord formation. These findings reveal a previously unappreciated role for macrophages in testis morphogenesis and suggest that macrophages are an intermediary between neovascularization and organ architecture during fetal organogenesis. Mammalian testis morphogenesis is a highly orchestrated process involving pre-Sertoli cells, germ cells, interstitial/mesenchymal CALML3 cells, and vascular endothelial cells (1), providing an ideal model system to study cellular interactions during fetal organ CL-387785 (EKI-785) patterning. In the mouse, cells in the XY (male) gonad undergo extensive cellular rearrangements between embryonic day (E) 11.5 and E12.5 that lead to the formation of testis cords, the precursors of seminiferous tubules in the adult organ (2). Pre-Sertoli cells express sex-determining genes, such as sex determining region of chromosome Y (and and Fig. S1are higher-magnification images of the boxed regions in and denote nascent gonads. At E10.5 (show independent macrophage markers. (points to a rare CD45+, IBA1? cell. (Scale bars: 50 m.) See also Figs. S1 and S2. Although and and and and and are higher-magnification images of the boxed regions in = 8 brains total), testis (= 16 testes), and liver (= 8 livers) from three independent litters. Colors of bars in correspond to the population whose percentages are labeled in the CL-387785 (EKI-785) same color in and are represented as means SEM. Lowercase letters (a versus b or y versus z) indicate statistically different values (< 0.05). See also Fig. S3. In the fetal liver, Tomato labeled a smaller percentage of F4/80+ cells and more F4/80? cells relative to the brain and testis (Fig. 2 and and and and and (19) relative to purified fetal liver macrophages (Fig. S3and were not [Fig. S3(33)]. Fetal Gonadal Macrophages Are M2-Like Macrophages in Active Cell Cycle. E12.5 and CL-387785 (EKI-785) and are higher-magnification images of the boxed regions in is MKI67 channel alone for the GFP+ cell indicated by the arrowhead), unlike adult testis macrophages (and arrowhead in are represented as means SEM. (in and are CD86- and MHCII-only channels, respectively, for macrophages indicated by arrowheads. (Scale bars: 50 m.) See also Figs. S4 and S5. Although we found that fetal gonad macrophages were in active cell cycle, we did not find any appreciable apoptosis of macrophages during fetal testis development. However, we did observe that activated (cleaved) Caspase-3+ cells were regularly engulfed by testis macrophages (Fig. S4 and and and also was reported recently to be enriched in yolk-sacCderived F4/80-bright macrophages (19), consistent with our lineage tracing results. These markers were expressed in distinct patterns in the fetal brain and liver (Figs. S2 and S5 and and and and and and and shows mesonephric ducts marked by diffuse SOX2 staining. (and and and in is a higher-magnification image of the coelomic vessel region CL-387785 (EKI-785) denoted by the arrowhead. (and indicates the normal formation of a coelomic vessel. Arrowheads in indicate dying vascular clumps in the absence of VEGF signaling. (and (was unaffected. +, 0.05<< 0.1; *< 0.05; **< 0.005. Also see Fig. S6. Testis macrophages expressed a number of endothelial markers including Neuropilin 1 (NRP1), which is a vascular endothelial growth factor (VEGF) coreceptor that normally is expressed in endothelial cells but also has been implicated as a marker of macrophages involved in neovascularization of tumors (36C38) (Fig. S6expression within differentiated macrophages. To investigate whether.