yolk-sac derived macrophages regulate fetal testis ... · yolk-sac–derived macrophages regulate...

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Yolk-sacderived macrophages regulate fetal testis vascularization and morphogenesis Tony DeFalco a,1 , Indrashis Bhattacharya a , Alyna V. Williams a , Dustin M. Sams a , and Blanche Capel b,1 a Division of Reproductive Sciences, Cincinnati Childrens Hospital Medical Center, Cincinnati, OH 45229; and b Department of Cell Biology, Duke University Medical Center, Durham, NC 27710 Edited by Janet Rossant, Hospital for Sick Children, University of Toronto, Toronto, Canada, and approved May 1, 2014 (received for review January 4, 2014) Organogenesis of the testis is initiated when expression of Sry 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 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 me- sonephric vasculature during the initial phases of testis morpho- genesis. Macrophages mediate vascular reorganization and prune errant germ cells and somatic cells after testis architecture is estab- lished. We show that gonadal macrophages are derived from primitive yolk-sac hematopoietic progenitors and exhibit hall- marks of M2 activation status, suggestive of angiogenic and tissue remodeling functions. Depletion of macrophages resulted in im- paired vascular reorganization and abnormal cord formation. These findings reveal a previously unappreciated role for macro- phages in testis morphogenesis and suggest that macrophages are an intermediary between neovascularization and organ architec- ture during fetal organogenesis. mononuclear phagocyte | myeloid cell | VEGF | endothelial cell | sex determination M ammalian testis morphogenesis is a highly orchestrated process involving pre-Sertoli cells, germ cells, interstitial/ mesenchymal cells, and vascular endothelial cells (1), providing an ideal model system to study cellular interactions during fetal organ 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 (Sry) and sex de- termining region Y (SRY)-box 9 (Sox9) (3, 4), and traditionally have been considered the main driving force in generating tes- ticular architecture. However, recent evidence from our laboratory and others suggests that endothelial and other interstitial mesenchymal cells also play critical roles in testis morphogenesis (58). Mononuclear phagocytes (MPs) represent a diverse subset of the myeloid immune cell lineage which includes macrophages and dendritic cells. MPs play diverse roles in developmental and disease contexts (9, 10), likely acting through their control of vessel anastomosis, clearing of apoptotic cells and other cellular debris, secretion of cytokines and growth factors, and modula- tion of extracellular matrix (9, 11). MPs are nearly ubiquitous in adult organs throughout the body and are involved in the mor- phogenesis of multiple tissues, such as bone, mammary gland ducts, pancreatic islets, and eye vasculature (1215). However, almost all these processes are postnatal tissue-remodeling events. The origin and function of MPs during fetal organogenesis is poorly understood. Traditional models assumed that hematopoietic stem cells (HSCs) differentiate into monocytes, which circulate through peripheral blood and subsequently are recruited to target tissues via local secretion of cytokines, leading to their differentiation into monocyte-derived lineages, e.g., granulocytes, dendritic cells, or macrophages (reviewed in ref. 10). Definitive hematopoiesis (and the appearance of HSCs) occurs initially in the yolk sac, placenta, and aorta-gonad-mesonephros region (AGM) at different time points between E8.5 and E10.5. HSCs populate the fetal liver shortly thereafter (E11.5E13.5), before the estab- lishment of the bone marrow. However, primitivehemato- poiesis, originating from unique yolk-sacderived progenitor cells, takes place at around E7.5, before definitive hematopoie- sis, and gives rise to hematopoietic cell types, including eryth- rocytes and macrophages, that migrate through the yolk-sac vasculature to colonize the fetus (16). Yolk-sacderived primi- tive macrophages can contribute to adult hematopoiesis and adult cell types (1719). In particular, lineage-tracing analyses showed that adult microglia (brain-specific macrophages) are derived exclusively from primitive yolk-sac precursors (17). Yolk- sacderived macrophages are distinct from HSC-derived mac- rophages: Yolk-sacderived macrophages are F4/80-bright, CD11b- dim, and myeloblastosis oncogene (Myb)- and FMS-like tyrosine kinase 3 (Flt3)-independent, whereas HSC-derived macrophages are F4/80-dim, CD11b-bright, and Myb- and Flt3-dependent (19). It is likely that the distinct origins of these cells impart unique identities and functions. In the postnatal and adult testis, macrophages make up a large portion of the interstitial compartment (20) and are important for Leydig cell development and steroidogenic function (21, 22). However, macrophages have not been detected or functionally characterized during fetal testis morphogenesis. We show that Significance A main requisite for organogenesis is the integration of vas- cular networks, which not only deliver oxygen and nutrients but also serve instructive roles in organ patterning that de- termine how organs develop into their final structure and function in the adult. Our previous work showed that vas- cularization is essential to induce formation of the primitive cord structures that give rise to the seminiferous tubules of the adult testis. Here we show that fetal macrophages are required to remodel the vasculature and refine organ com- partments during differentiation of the gonad. This study reveals an underappreciated and likely vital role for mac- rophages in fetal organogenesis that may be relevant to the development of many organs. Author contributions: T.D. and B.C. designed research; T.D., I.B., A.V.W., and D.M.S. per- formed research; T.D., I.B., and B.C. analyzed data; and T.D. and B.C. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1400057111/-/DCSupplemental. E2384E2393 | PNAS | Published online May 27, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1400057111 Downloaded by guest on November 6, 2020

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Page 1: Yolk-sac derived macrophages regulate fetal testis ... · Yolk-sac–derived macrophages regulate fetal testis vascularization and morphogenesis Tony DeFalcoa,1, Indrashis Bhattacharyaa,

Yolk-sac–derived macrophages regulate fetal testisvascularization and morphogenesisTony DeFalcoa,1, Indrashis Bhattacharyaa, Alyna V. Williamsa, Dustin M. Samsa, and Blanche Capelb,1

aDivision of Reproductive Sciences, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; and bDepartment of Cell Biology, Duke UniversityMedical Center, Durham, NC 27710

Edited by Janet Rossant, Hospital for Sick Children, University of Toronto, Toronto, Canada, and approved May 1, 2014 (received for review January 4, 2014)

Organogenesis of the testis is initiated when expression of Sry inpre-Sertoli cells directs the gonad toward a male-specific fate. Thecells in the early bipotential gonad undergo de novo organizationto form testis cords that enclose germ cells inside tubules lined byepithelial Sertoli cells. Although Sertoli cells are a driving force inthe de novo formation of testis cords, recent studies in mouseshowed that reorganization of the vasculature and of interstitialcells also play critical roles in testis cord morphogenesis. However,the mechanism driving reorganization of the vasculature duringfetal organogenesis remained unclear. Here we demonstrate thatfetal macrophages are associated with nascent gonadal and me-sonephric vasculature during the initial phases of testis morpho-genesis. Macrophages mediate vascular reorganization and pruneerrant germ cells and somatic cells after testis architecture is estab-lished. We show that gonadal macrophages are derived fromprimitive yolk-sac hematopoietic progenitors and exhibit hall-marks of M2 activation status, suggestive of angiogenic and tissueremodeling functions. Depletion of macrophages resulted in im-paired vascular reorganization and abnormal cord formation.These findings reveal a previously unappreciated role for macro-phages in testis morphogenesis and suggest that macrophages arean intermediary between neovascularization and organ architec-ture during fetal organogenesis.

mononuclear phagocyte | myeloid cell | VEGF | endothelial cell |sex determination

Mammalian testis morphogenesis is a highly orchestratedprocess involving pre-Sertoli cells, germ cells, interstitial/

mesenchymal cells, and vascular endothelial cells (1), providingan ideal model system to study cellular interactions during fetalorgan patterning. In the mouse, cells in the XY (male) gonadundergo extensive cellular rearrangements between embryonicday (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 sexdetermining region of chromosome Y (Sry) and sex de-termining region Y (SRY)-box 9 (Sox9) (3, 4), and traditionallyhave been considered the main driving force in generating tes-ticular architecture. However, recent evidence from ourlaboratory and others suggests that endothelial and otherinterstitial mesenchymal cells also play critical roles in testismorphogenesis (5–8).Mononuclear phagocytes (MPs) represent a diverse subset of

the myeloid immune cell lineage which includes macrophagesand dendritic cells. MPs play diverse roles in developmental anddisease contexts (9, 10), likely acting through their control ofvessel anastomosis, clearing of apoptotic cells and other cellulardebris, secretion of cytokines and growth factors, and modula-tion of extracellular matrix (9, 11). MPs are nearly ubiquitous inadult organs throughout the body and are involved in the mor-phogenesis of multiple tissues, such as bone, mammary glandducts, pancreatic islets, and eye vasculature (12–15). However,almost all these processes are postnatal tissue-remodeling events.The origin and function of MPs during fetal organogenesis is

poorly understood. Traditional models assumed that hematopoietic

stem cells (HSCs) differentiate into monocytes, which circulatethrough peripheral blood and subsequently are recruited totarget tissues via local secretion of cytokines, leading to theirdifferentiation into monocyte-derived lineages, e.g., granulocytes,dendritic cells, or macrophages (reviewed in ref. 10). Definitivehematopoiesis (and the appearance of HSCs) occurs initially in theyolk sac, placenta, and aorta-gonad-mesonephros region (AGM)at different time points between E8.5 and E10.5. HSCs populatethe fetal liver shortly thereafter (E11.5–E13.5), before the estab-lishment of the bone marrow. However, “primitive” hemato-poiesis, originating from unique yolk-sac–derived progenitorcells, takes place at around E7.5, before definitive hematopoie-sis, and gives rise to hematopoietic cell types, including eryth-rocytes and macrophages, that migrate through the yolk-sacvasculature to colonize the fetus (16). Yolk-sac–derived primi-tive macrophages can contribute to adult hematopoiesis andadult cell types (17–19). In particular, lineage-tracing analysesshowed that adult microglia (brain-specific macrophages) arederived exclusively from primitive yolk-sac precursors (17). Yolk-sac–derived macrophages are distinct from HSC-derived mac-rophages: Yolk-sac–derived macrophages are F4/80-bright, CD11b-dim, and myeloblastosis oncogene (Myb)- and FMS-like tyrosinekinase 3 (Flt3)-independent, whereas HSC-derived macrophagesare F4/80-dim, CD11b-bright, andMyb- and Flt3-dependent (19).It is likely that the distinct origins of these cells impart uniqueidentities and functions.In the postnatal and adult testis, macrophages make up a large

portion of the interstitial compartment (20) and are importantfor Leydig cell development and steroidogenic function (21, 22).However, macrophages have not been detected or functionallycharacterized during fetal testis morphogenesis. We show that

Significance

A main requisite for organogenesis is the integration of vas-cular networks, which not only deliver oxygen and nutrientsbut also serve instructive roles in organ patterning that de-termine how organs develop into their final structure andfunction in the adult. Our previous work showed that vas-cularization is essential to induce formation of the primitivecord structures that give rise to the seminiferous tubules ofthe adult testis. Here we show that fetal macrophages arerequired to remodel the vasculature and refine organ com-partments during differentiation of the gonad. This studyreveals an underappreciated and likely vital role for mac-rophages in fetal organogenesis that may be relevant to thedevelopment of many organs.

Author contributions: T.D. and B.C. designed research; T.D., I.B., A.V.W., and D.M.S. per-formed research; T.D., I.B., and B.C. analyzed data; and T.D. and B.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. E-mail: [email protected] [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1400057111/-/DCSupplemental.

E2384–E2393 | PNAS | Published online May 27, 2014 www.pnas.org/cgi/doi/10.1073/pnas.1400057111

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yolk-sac–derived macrophages are the only major myeloidcell type in the fetal gonad during morphogenesis of thetestis. These macrophages interacted with multiple cell typesbut were prominent near developing vasculature. Specificdepletion of macrophages resulted in significant vascular andarchitectural abnormalities in the fetal testis. These studiesuncover a previously unidentified role for gonadal–meso-nephric macrophages in testis morphogenesis, consistent witha broader role for macrophages in fetal development andorganogenesis.

ResultsMacrophages Constitute the Major Myeloid Cell Type Present in theGonad–Mesonephros Primordium During Initial Testis Differentiation.To label macrophages during the initial stages of gonad forma-tion, we used a well-characterized macrophage cell-surface marker,F4/80 (20, 23). At E9.5, before gonad specification, we did notdetect macrophages in the presumptive gonadal region or alongthe migratory path germ cells travel to the urogenital ridge (UGR)(Fig. S1A). However, we detected numerous F4/80+ cells inthe brain, consistent with previous reports of early yolk-sac–derived macrophages entering the head before that time (17,19) (Fig. S1B). By E10.5, we detected F4/80+ cells in the brain(Fig. 1A) as well as in the UGR, which includes the gonad–mesonephros primordium (Fig. 1B). F4/80+ cells had a stellatemorphology characteristic of macrophages, strongly expressedchemokine (C-X3-C motif) receptor 1 (Cx3cr1)-GFP at allstages examined (24) (Fig. 1 C and D and Fig. S1C), and wereconcentrated in the mesonephros near the gonad border atE10.5 (Fig. 1 B–D).Although Cx3cr1-GFP is expressed in multiple immune cell

types in adult tissues (24), flow cytometry experiments revealedthat >95% of Cx3cr1-GFP+ fetal gonad cells were double-posi-tive for the macrophage markers F4/80 and CD11b (25) (Fig.S1D), demonstrating that Cx3cr1-GFP is an effective and spe-cific marker of gonadal–mesonephric macrophages during fetalstages. Quantitative RT-PCR (qPCR) analysis of purified GFP+

gonadal–mesonephric cells obtained via fluorescence-activatedcell sorting (FACS) also showed a significant (∼1,000-fold) en-richment of the macrophage markers EGF-like module con-taining, mucin-like, hormone receptor-like sequence 1 (Emr1)(which encodes F4/80) and colony stimulating factor 1 receptor(Csf1r) (also called “c-fms”) (26) relative to GFP− cells (Fig. S1E and F).

At E11.5, F4/80- and Cx3cr1-GFP–expressing cells near thegonad border increased in number and also expressed CSF1Rand the microglial/macrophage marker ionized calcium bindingadaptor molecule 1 (IBA1) (also called “AIF1”) (27, 28) (Fig. 1 Eand F), consistent with a macrophage identity. Macrophages werefound occasionally within the gonad at this stage but still wereconcentrated near the gonad–mesonephros boundary at E11.5and E12.5 (Fig. 1 E–G).At E12.5, ∼90% of cells positive for the pan-leukocyte marker

CD45 coexpressed IBA1 by immunofluorescence (Fig. 1H),suggesting that macrophages are the major MP cell type present.Flow cytometry analysis of gonadal CD45+ cells revealed a minorMP population (9.2% of CD45+ cells) immunoreactive for Gr-1(Fig. S2A), a marker for granulocytes such as neutrophils (29).We saw virtually no immunoreactivity via flow cytometry (3.1%of CD45+ cells) for the dendritic cell marker CD11c (also called“ITGAX”) (30) (Fig. S2A), in contrast to hematopoietic fetalliver, where higher numbers of both CD45/CD11c+ dendriticcells (25.9%) and CD45/Gr-1+ granulocytes (16.5%) were pres-ent (Fig. S2B). Finally, immunofluorescence analyses showedvirtually no lymphoid cells (B cells or T cells) present in thegonad during initial testis differentiation (Fig. S2 C–J).

Gonadal–Mesonephric Macrophages Arise from Primitive Yolk-Sac–Derived Hematopoietic Progenitors. To determine whether gonadal–mesonephric macrophages are derived from the yolk sac, wedelivered a pulse of 4-hydroxytamoxifen at E7.5 to induce ac-tivity of Csf1r-CreER [which is active in yolk-sac cells at thisstage (31)] and to label primitive yolk-sac progenitors perma-nently with a Cre-responsive Rosa-Tomato red fluorescent line-age tracer (32). We subsequently assessed Tomato+ cells at E14.5,a stage when tissue-resident macrophages are readily distinguish-able as being yolk-sac–derived or HSC-derived by their relativelevels of F4/80 and CD11b expression (19). As a positive controlwe examined fetal brains, which are colonized by primitive yolk-sac–derived macrophages at around E8.5–E9.5 (17). As expected,we detected extensive Tomato labeling throughout the brain,which colocalized with F4/80 (Fig. 2A). Flow cytometry confirmedthat Tomato efficiently labeled F4/80+ and CD11b+ macrophages(Fig. 2 G–I). We did not detect any Tomato labeling of Cre− lit-termate controls in any organs we assayed (Fig. 2 B, D, and F).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 C and I). Flow cytometry confirmed this result and revealed

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Fig. 1. Macrophages are present in the fetal gonad primordium during initial gonad formation. Immunofluorescent images of E10.5 brain (A), E10.5 UGRs(B–D), and E11.5-E12.5 XY gonads (E–H). In all figures, colors of immunofluorescent markers are indicated. White dashed outlines indicate the gonad–mesonephros border in all figures. A′, B′, and G′ are higher-magnification images of the boxed regions in A, B, and G, respectively. Yellow dashed outlines in Band C denote nascent gonads. At E10.5 (A–D), F4/80+ macrophages were present in the brain and the UGR. Macrophages in the E10.5 UGR also expressedCx3cr1-GFP (C) and were concentrated near the gonad (g)–mesonephros (m) border (D). (E–G) At E11.5, gonads contained IBA1+, Cx3cr1-GFP+, CSF1R+, andF4/80+ macrophages near the gonad–mesonephros border. GATA4 labels gonadal somatic cells. In F′, single-channel versions of the boxed region in F showindependent macrophage markers. (H) CD45 staining revealed extensive colocalization with IBA1. The arrowhead in H points to a rare CD45+, IBA1− cell.(Scale bars: 50 μm.) See also Figs. S1 and S2.

DeFalco et al. PNAS | Published online May 27, 2014 | E2385

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a marked increase in the number of CD11b-bright cells (as op-posed to CD11b-intermediate cells) in the liver (Fig. 2 G and H).This result is consistent with an enrichment of HSC-derived mac-rophages in the liver (19), suggesting a more complex origin for fetalliver macrophages. kit oncogene (C-KIT), a marker of HSCs,stained some Tomato+ cells in the fetal liver, but many were C-KIT−, perhaps suggesting they had differentiated and no longerexpressed C-KIT (Fig. S3 A and B).In the fetal testis, Tomato labeling was extensive and overlapped

with F4/80+ cells (Fig. 2E). Flow cytometry revealed that the ex-pression profiles of testis and brain macrophages were virtually

identical, with an enrichment of F4/80-bright, CD11b-intermediatecells (Fig. 2G andH), consistent with yolk-sac–derived macrophagesrather than HSC-derived macrophages (19). This result suggeststhat, like brain microglia (17), gonadal macrophages are derivedalmost exclusively from primitive yolk-sac progenitors, with anegligible contribution of HSC-derived cells that arise later indevelopment from the yolk sac, AGM, or placenta.Consistent with this hypothesis, purified gonadal macrophages

expressed lower levels of the HSC-associated genes Myb and Flt3(19) relative to purified fetal liver macrophages (Fig. S3C).These data agree with our previous gonad microarray analysis

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Fig. 2. Gonadal macrophages arise from primitive yolk-sac–derived progenitors. (A–F) Immunofluorescence images of fetal brains (A and B), livers (C and D),and testes (E and F) from E12.5 Csf1r-CreER; Rosa-Tomato (A, C, and E) and littermate control embryos carrying Rosa-Tomato, but no Cre recombinase (B, D,and F) injected at E7.5 with 4-hydroxytamoxifen. A′–F′ are higher-magnification images of the boxed regions in A–F, respectively. Expression of Tomato redfluorescent protein was detected only in Csf1r-CreER; Rosa-Tomato embryos (A, C, and E) but not in control littermates (B, D, and F) exposed to 4-hydrox-ytamoxifen in utero. Tomato-positive macrophages (A′, C′, and E′, arrowheads) were observed in all three organs. Occasionally, Tomato was observed in F4/80− cells in gonads and other tissues (arrows). (Scale bars: 50 μm.) (G) Flow cytometric analysis of Tomato+ cells from a representative litter of E14.5 Csf1r-CreER; Rosa-Tomato embryos injected at E7.5 with 4-hydroxytamoxifen. Tomato+ cells from fetal brain (Left) and testis (Center) contained mostly F4/80-high,CD11b-intermediate cells, but livers (Right) showed a shift toward F4/80-intermediate, CD11b-high cells. Numbers next to polygonal gates indicate thepercent of Tomato+ cells within the adjacent gate. (H) Graph shows the percent of Tomato+ cells expressing F4/80 and CD11b in XY brain (n = 8 brains total),testis (n = 16 testes), and liver (n = 8 livers) from three independent litters. Colors of bars in H correspond to the population whose percentages are labeled inthe same color in G. (I) Graph shows percent efficiency of Tomato labeling in F4/80+ cells by flow cytometry in E14.5 brain, testis, and liver in the littersanalyzed in H. Data in H and I are represented as means ± SEM. Lowercase letters (a versus b or y versus z) indicate statistically different values (P < 0.05). Seealso Fig. S3.

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(33), in which the yolk-sac macrophage–associated gene spleenfocus forming virus proviral integration oncogene (Sfpi1; alsocalled “Pu.1”) was detected in hematopoietic-derived cells butthe HSC-associated genes Myb and Flt3 were not [Fig. S3D (33)].

Fetal Gonadal Macrophages Are M2-Like Macrophages in Active CellCycle. E12.5 Cx3cr1-GFP gonadal macrophages were nearly100% positive for a marker of active cell cycle, antigen identifiedby monoclonal antibody Ki 67 (MKI67) (Fig. 3A), and somemacrophages expressed the M-phase marker phospho-histoneH3 (pHH3) (Fig. S4 A and B). These data are consistent with theidea that, once populated from yolk-sac progenitors, fetal mac-rophages proliferate locally to increase their numbers ratherthan recruiting from other hematopoietic sources such as theAGM. Adult testes, in contrast to fetal testes, containedmacrophages that were MKI67− (Fig. 3B), indicating that cer-tain cell-cycle properties are unique to fetal or adult testicularmacrophages.Although we found that fetal gonad macrophages were in

active cell cycle, we did not find any appreciable apoptosis ofmacrophages during fetal testis development. However, we didobserve that activated (cleaved) Caspase-3+ cells were regularly

engulfed by testis macrophages (Fig. S4 C and D; see also be-low). Consistent with our MKI67 and pHH3 data suggesting thatmacrophages proliferate in situ and show negligible cell death,we found a steady increase in the number of macrophages ob-served in the fetal testis between E10.5 and E13.5 (Fig. 3C).Macrophages are characterized by M1/M2 activation status,

defined by specific cell-surface markers and by the secretion ofa characteristic subset of proinflammatory or anti-inflammatorycytokines (34). M1 macrophages traditionally are associated withproinflammatory, cytotoxic responses, whereas M2 macrophagesare anti-inflammatory and are linked to tissue remodeling andangiogenesis. The expression of two markers of M1 macro-phages, CD86 and major histocompatibility complex, class II(MHCII), was very low based on fluorescent antibody staining offetal testes (Fig. 3 D and E). In contrast, two M2 macrophagemarkers, CD206 (also called “MRC1”) and avian muscu-loaponeurotic fibrosarcoma AS42 oncogene homolog (C-MAF)(34), were strongly expressed in virtually 100% of IBA1+ and F4/80+ cells (Fig. 3 F and G). C-MAF is a transcription factor thatmediates the immunosuppressive activity of the M2-associatedanti-inflammatory cytokine interleukin 10 (IL10) (35), and itsexpression in macrophages is consistent with an M2 status. c-Mafalso was reported recently to be enriched in yolk-sac–derivedF4/80-bright macrophages (19), consistent with our lineagetracing results. These markers were expressed in distinct patternsin the fetal brain and liver (Figs. S2 and S5 A–D): Similar togonadal macrophages, brain macrophages were M2-like, butfetal liver macrophages were more M1-like, with stronger ex-pression of CD86 and weak expression of CD206 (Fig. S5 A–D).In addition, qPCR analysis of FACS-purified Cx3cr1-GFP cellsfrom E12.5 XY brains, gonads, and livers revealed that, similarto brain macrophages and in contrast to liver macrophages, go-nadal macrophages expressed lower levels of the M1-associatedgene interleukin 12 (Il12) and higher levels of the M2-associatedgene arginase 1 (Arg1) (Fig. S5 E and F). All these results suggestthat gonadal macrophages are M2-like, a state that is associatedwith vascular and tissue remodeling (11, 36).

Fetal Gonadal Macrophages Are Localized near DevelopingVasculature in the Nascent Testis. By E10.5, macrophages werelocalized specifically to the developing vascular plexus along thegonad–mesonephros border and to vascular branches betweenthe mesonephric ducts (Fig. 4 A and B). Between E11.5 andE13.5, the peak of vascular remodeling and neovascularization ofthe testis, large numbers of macrophages accumulated in thegonad–mesonephric region (Fig. 4 C–G). They were located nearthe vascular plexus and along the coelomic surface artery and itsassociated side branches, often wrapping long processes aroundendothelial cells (Fig. 4 F and G).Testis macrophages expressed a number of endothelial mark-

ers including Neuropilin 1 (NRP1), which is a vascular endo-thelial growth factor (VEGF) coreceptor that normally isexpressed in endothelial cells but also has been implicated asa marker of macrophages involved in neovascularization oftumors (36–38) (Fig. S6A); endothelial-specific receptor tyrosinekinase (TEK; also called “TIE2”) (Fig. S6B), an Angiopoietinreceptor expressed in macrophages associated with tumor neo-vascularization (36–38); and Tie2-Cre (Fig. S6C), as we pre-viously reported using a Cre-responsive GFP reporter strain (39).Tie2-Cre is not expressed in hematopoietic cells of the yolk sac (40),suggesting that the expression of Tie2-Cre is not merely the result ofthe labeling of early hematopoietic progenitors but reflects denovo Tie2 expression within differentiated macrophages.To investigate whether macrophages are principal recruiters of

endothelial cells, we used Cx3cr1-GFP to FACS-purify macro-phages and determined their expression of three proangiogenicfactors, vascular endothelial growth factor A (Vegfa), angio-poietin 1 (Angpt1), and angiopoietin 2 (Angpt2) relative to the

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Fig. 3. Fetal gonadal macrophages are in active cell cycle and are in M2activation status. Immunofluorescence images of E12.5 fetal (A and D–G),and adult (B) testes. A′, B′, and D′–G′ are higher-magnification images of theboxed regions in A, B, and D–G, respectively. Fetal gonadal macrophages (A)are positive for MKI67 (arrowhead in A′; Inset is MKI67 channel alone for theGFP+ cell indicated by the arrowhead), unlike adult testis macrophages(B and arrowhead in B′). CD68 marks macrophages in B. (C) Graph shows thenumber of F4/80+ macrophages per single 450 × 336 μm confocal opticalsection in the testis–mesonephros region of E10.5–E13.5 wild-type CD-1embryos (each stage contains ≥12 gonads). Data in C are represented asmeans ± SEM. (D–G) Fetal testis macrophages show low expression levels ofthe M1-associated markers CD86 (D) and MHCII (E) but are strongly positivefor the M2-associated markers CD206 (F) and C-MAF (G). C-MAF also isexpressed in interstitial cells of the fetal gonad (8). Insets in D′ and E′ areCD86- and MHCII-only channels, respectively, for macrophages indicated byarrowheads. (Scale bars: 50 μm.) See also Figs. S4 and S5.

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GFP− population of the gonad and mesonephros. GFP+ mac-rophages expressed detectable, albeit low, levels of Vegfa andAngpt1 but not of Angpt2 (Fig. S6D). However, macrophageexpression of Vegfa (and Angpt1) was lower than that of thenonmacrophage cell populations of the gonad/mesonephros(Fig. S6 D and E), suggesting, in agreement with our previousreport (7), that macrophage-derived VEGF is not the majorsource of VEGF in the gonad. By immunofluorescence, wedetected bright VEGFA punctae within gonadal macrophages(Fig. S6F). These punctae could represent autonomously pro-duced VEGFA; alternatively, macrophages might sequester orrelay VEGFA from the environment.

Gonad Macrophage Number and Localization Require Vasculature.Given our observation that testis macrophages are found con-sistently in close proximity to blood vessels, we hypothesized thatvasculature is required for the maintenance, recruitment, ormigration of fetal testis macrophages. To test this hypothesis, wedepleted testis vasculature in our whole-organ culture system(41) using a VEGF receptor small-molecule inhibitor, VEGFRtyrosine kinase inhibitor (TKI) II (5) and observed the effects onmacrophage number and gene expression. We achieved robustvascular depletion, visualized via dramatically reduced anti-platelet/endothelial cell adhesion molecule 1 (PECAM1) endo-thelial cell immunofluorescence (Fig. 4 H and I), and a 90–97%reduction in cadherin 5 (Cdh5) (also called “VE-Cad”) testisexpression (relative to control cultured littermate testes) viaqPCR (Fig. 4L). These effects were observed in fetal testescultured at both E11.5 and E12.5. We saw no evidence of apo-ptosis or reduction in germ cells based on PECAM1 and cleavedCaspase-3 staining (Fig. 4 H and I). Consistent with this result,the expression of germ cell [POU domain, class 5, transcriptionfactor 1 (Pou5f1); also called “Oct4”] and Sertoli cell (Sox9)markers was unchanged based on qPCR (Fig. 4L). These dataindicate that overall tissue health was maintained in vascular-depleted organ explants.

When we assessed macrophage number and gene expressionin vascular-depleted testes, we saw that, although macrophagenumber in the overall UGR (gonad plus mesonephros) was un-affected, there were fewer macrophages in vascular-depletedgonads, particularly in regions where the newly formed coelomicvasculature normally would reside (Fig. 4K). By qPCR, we sawa 55–65% reduction in both Emr1 (F4/80) and Csf1r expression(Fig. 4L) in the testis, although this effect was more significantwhen gonads were cultured at E12.5 than at E11.5. This greaterreduction likely occurs because more macrophages are normallypresent in the gonad by E12.5 (Fig. 4 C and E), so a reduction ingonad-specific macrophage number or gene expression uponvascular depletion is more robust at later stages. These datasuggest that macrophage localization and/or migration into thegonad are dependent on VEGF-mediated vascularization of thefetal testis.

Gonadal Macrophages Promote Tissue Clearance and Remodelingto Regulate Morphogenesis. In gonad development, germ cellssometimes are lost in the mesonephros en route to the gonad.Although it was assumed that these cells die by apoptosis, ouranalyses revealed that, at E11.5 and E12.5, germ cells remaining inthe mesonephros tended to cluster and nearly always were asso-ciated with or engulfed by macrophages: We observed SOX2+

nuclei completely contained within F4/80+ cells (Fig. 5A). Thesegerm cells were apoptotic, because we often observed C-KIT+/Caspase-3+ cells engulfed by macrophages (Fig. 5B). These resultssuggest that macrophages are involved in the clearance of mis-located germ cells during initial gonad formation.In normal gonad morphogenesis, Sertoli cells, like germ cells,

are rarely found outside testis cords. At E12.5, when testis cordshave formed completely, we observed macrophages within theinterstitial compartment completely surrounding anti-Müllerianhormone (AMH)-positive cellular material (Fig. 5C), suggestingthat macrophages also engulf or phagocytize AMH-expressingSertoli cells that fail to be incorporated into cords during initialtestis morphogenesis.

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Fig. 4. Fetal macrophages are intimately associated with nascent vasculature in the developing testis. Immunofluorescence images of E10.5 (A and B), E11.5(C and D), E12.5 (E and F), E13.5 (G) fetal testes, and E11.5 (H–K) cultured fetal testes. (A and B) At E10.5 a vascular plexus (vp, yellow bracket in A) already isvisible between the GATA4+ gonad (containing SOX2+ germ cells) and the neighboring mesonephros, and fetal macrophages concentrated near nascentvasculature are starting to invade the gonad (arrows in B). The asterisk in A shows mesonephric ducts marked by diffuse SOX2 staining. (C and D) By E11.5,macrophages are enriched in the vascular plexus (vp) near the gonad–mesonephros border and near vascular sprouts in the gonad (arrows in D). Kdr-mCherrylabels endothelial cells (42). (E–G) By E12.5 (E and F) and E13.5 (G), an increase in the number of macrophages is evident, concentrated in the vascular bed atthe gonad border and near the newly formed testis vasculature (arrowheads in E and G). Inset in G is a higher-magnification image of the coelomic vesselregion denoted by the arrowhead. (H and I) Relative to control E11.5 cultured testes (H), vascular-depleted testes cultured in VEGFR TKI II (I) do not show anincrease in apoptosis of nonendothelial cells. The arrow in H indicates the normal formation of a coelomic vessel. Arrowheads in I indicate dying vascularclumps in the absence of VEGF signaling. (J and K) Vascular-depleted testes show altered localization of macrophages relative to controls. (Scale bars: 50 μm.)(L) The graph shows fold change in gene expression in E11.5 and E12.5 vascular-depleted testes cultured in VEGFR TKI II (removed from mesonephros) relativeto cultured control testes for markers of germ cells (Oct4), Sertoli cells (Sox9), endothelial cells (Cdh5), and macrophages [Emr1 (F4/80) and Csf1r]. Expressionof Vegfa was unaffected. +, 0.05<P < 0.1; *P < 0.05; **P < 0.005. Also see Fig. S6.

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During the stages when extensive vascular remodeling is tak-ing place in the XY gonad (E11.5–E12.5), we often observedkinase insert domain protein receptor (Kdr)-mCherry+ (alsoknown as “Flk” or “Vegfr2”) endothelial cells (42) completelyengulfed by macrophages, both in the mesonephric vascularplexus and in the surface testis vasculature (Fig. 5 D and E). Wecommonly observed apoptotic endothelial cells, especially withinthe mesonephric vascular plexus (Fig. 5F), and these dying en-dothelial cells (PECAM1+/Caspase-3+ cells) also were engulfedby macrophages (Fig. 5G). Kdr-mCherry+ material containedwithin macrophages colocalized with CD68 (also called “mac-rosialin”), a marker of macrophage-specific lysosomes and endo-somes of the phagocytic pathway (43, 44) (Fig. 5H), suggestingthat macrophages may physically break down the vascular plexusto free individual endothelial cells, which then can migrate fromthe mesonephros into the gonad (39).Although the localization of macrophages is interstitium-

specific at postnatal stages (20), we found a large number of mac-rophages within testis cords in the fetal gonad (Fig. 5I). Thesemacrophages had a much more stellate morphology than theirinterstitial counterparts and were intercalated between Sertolicells and germ cells, making contact with multiple cells in thecord. Localization within cords was observed only during fetalstages. By birth, macrophages were restricted to the interstitiumand were excluded from testis cords (Fig. S7), similar to theirlocalization in adult testes (20).

Macrophages Are Required for Vascular Breakdown and Testis CordMorphogenesis. To determine whether macrophages play a criti-cal role in vascularization and testis morphogenesis, we used theRosa-eGFP-Diphtheria Toxin A (Rosa-eGFP-DTA) system (45),under the control of Cx3cr1-Cre to deplete macrophages spe-cifically. To confirm that Cx3cr1-Cre is active in early macro-phages, we used a Cre-responsive farnesylated-GFP (Rosa-fGFP)

reporter (46) as a lineage tracer and fluorescent reporter todetect Cre-expressing cells. At E10.5, GFP+ cells were detectedin double-positive embryos within the brain, yolk sac, and UGR,and ∼95% of these cells expressed F4/80 (Fig. 6 A–C), whereasCre− littermates showed no GFP expression (Fig. 6D). Similarresults were obtained using an independent Cre-responsive Rosa-Tomato lineage tracer (32) (Fig. 6E). In these experiments, Tomatowas not detected in Sertoli cells, germ cells, or vasculature(Fig. 6F), demonstrating that Cx3cr1-Cre is an effective andspecific Cre line for targeting fetal macrophages.E13.5 Cx3cr1-Cre; Rosa-eGFP-DTA (macrophage-depleted)

embryos showed no significant gross anatomical abnormalitiesand were recovered in expected numbers from litters at E13.5.The Cx3cr1-Cre; Rosa-eGFP-DTA system consistently exhibitednearly complete ablation (∼95%) of macrophages (Fig. 6 G–I).Although a robust, coherent coelomic artery was present incontrols, fewer endothelial cells migrated into the coelomic re-gion of the macrophage-depleted E13.5 Cx3cr1-Cre; Rosa-eGFP-DTA gonads, and the coelomic vessel was poorly organized withfewer endothelial cells (Fig. 7 A–D). Reciprocally, the meso-nephric vascular plexus was enlarged in depleted animals andshowed a dramatic increase in the overall vasculature remainingin the mesonephros (Fig. 7 E–H).Wild-type E13.5 testes had well-established cords that were of

regular size and spacing (Fig. 7I). Although gross partitioningof interstitial cells from Sertoli and germ cells took place inmacrophage-ablated samples, cords were irregularly branchedand fused and often failed to reach the coelomic surface regionof the gonad (Fig. 7J). There was some variability in the severityof cord architecture defects, although abnormalities were consis-tent. Interestingly, small clusters of three to five Sertoli cells weredetected outside the boundaries of testis cords (Fig. 7J, Inset),consistent with an absence of the surveillance function of testismacrophages.

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Fig. 5. Fetal gonadal macrophages play roles in tissue clearance and remodeling to regulate tissue morphogenesis. Immunofluorescence images of E11.5(A and D) and E12.5 (B, C, and E–I) fetal testes. A′–F′ are higher-magnification images of the boxed regions in A–F, respectively. (A) At E11.5, SOX2+ germ cellsin the mesonephros are engulfed by macrophages. A number of these engulfed germ cells (C-KIT+) are apoptotic (cleaved Caspase-3+ cells; arrowheads in B′).Panels to right of B′ are Caspase-3-only and C-KIT-only channels for the image in B′. (C and C′) AMH+ Sertoli cells remaining in the interstitium (i) outside thetestis cords (tc) are surrounded by macrophages. Dashed outlines in C′ denote testis cord boundaries. (D and E) Macrophages (arrowheads in D′) surroundvascular sprouts in the region of the gonad–mesonephros border (dashed line in D) as well as in gonad surface vasculature (arrowheads in E). The Inset toright of E′ is a higher-magnification image of the cell denoted by the open arrowhead in E indicating internalized vascular material. (F and G) A number ofapoptotic PECAM1+ endothelial cells (F and F′) are engulfed by Cx3cr1-GFP+ macrophages (G). (H) Engulfed vascular material is colocalized with macrophagephagolysosomal marker CD68 within a NRP1+ macrophage (outlined). (I) By E12.5 macrophages also are within testis cords (closed arrowheads). The Insets tothe right of I are higher-magnification images of the macrophage–germ cell interaction denoted by open arrowhead in I. (Thin scale bars: 50 μm; thick scalebars: 12.5 μm.) Also see Fig. S7.

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Although the distribution of vasculature was abnormal inmacrophage-depleted testes, the overall number of endothelialcells was grossly unaffected. To assess the direct effects ofmacrophage depletion on testis cell types, and on endothelialcells in particular, we examined cell-cycle activity (by MKI67 andpHH3 expression) and apoptosis (by cleaved Caspase-3 expres-sion) in macrophage-depleted testes. In Cx3cr1-Cre; Rosa-DTAfetal testes we saw normal expression of MKI67 and pHH3in PECAM1+ endothelial cells (Fig. 7 K–N). We also noticed nodramatic increase or decrease of Caspase-3 expression within thevasculature or in the testis overall (Fig. 7 O and P). We assumethat the few macrophages remaining after depletion are suffi-cient to maintain a level of cell clearance required by the lowlevel of cell death that occurs during testis development (see Fig.4H). Overall, these data suggest that fetal testis macrophages are

required for vascular remodeling but not for cell-cycle activity orsurvival of endothelial cells.

DiscussionThe Presence of Macrophages in the Fetal Testis. Although macro-phages are known to populate the interstitium of the postnataland adult rat testis (20, 47), little is known about their fetalorigins. Although some reports have documented the presenceof macrophages in the fetal rat testis as early as E16.5–E19 (47,48), there has been no investigation of the role of macrophagesduring the initial steps of fetal testis organogenesis. Here wedemonstrated that differentiated macrophages expressing well-characterized markers such as F4/80 and IBA1 are detectable inthe UGR at the initial stages of gonad specification (E10.5).Because our data suggested that the great majority of macro-phages during initial gonad differentiation (E10.5–E12.5) arelocalized to the mesonephric region near the gonad border, it ispossible that macrophages were not detected in previous studiesbecause the mesonephros was not examined. However, a sub-stantial increase in numbers of macrophages also was observedwithin the gonad by E12.5–E13.5, likely because of the pro-liferation of resident cells. Testicular macrophages have beenreported in many diverse nonmammalian species, includingswan, catfish, and lizard (49–51). Thus, it is possible that mac-rophages are present in the nascent gonad in all vertebrates andmay have evolutionarily conserved roles.

Yolk-Sac–Derived Primitive Macrophages Play Diverse Roles in Fetaland Adult Organogenesis. Traditional models of hematopoiesis arecentered around the dogma that HSCs give rise to precursorsthat patrol the peripheral blood and enter tissues that secretemacrophage recruitment or differentiation factors (16). Althoughthis model of definitive HSC-based hematopoiesis has dominatedthe fields of immunology and hematology for decades, recentlineage-tracing experiments have revealed that primitive hemato-poiesis in the yolk sac plays a significant role in both fetal andadult myelopoiesis (17–19).In this study, we demonstrated that primitive yolk-sac hema-

topoietic progenitors give rise to gonadal–mesonephric macro-phages. This result was surprising, given the proximity of theUGR to the AGM region, where a subset of definitive HSCprogenitors arises. Other groups have shown that a subset ofother organ-specific macrophages (19) and microglia in the brain(17) also are derived exclusively from primitive yolk-sac pro-genitors. Although our data are consistent with a yolk-sacprimitive hematopoietic origin for gonad macrophages, we can-not formally rule out the possibility that a subset of gonadmacrophages also is derived from the yolk sac at later stages, orfrom other sites of definitive hematopoiesis such as the AGM orplacenta. However, we observed that the efficiency of tamoxifenlabeling with Csf1r-CreER;Rosa-Tomato is virtually identicalin gonad macrophages and brain macrophages (microglia), thelatter of which are derived exclusively from yolk-sac–derivedprimitive hematopoietic progenitors (18). If a subset of gonadmacrophages comprised definitive macrophages arising later indevelopment (i.e., at E8.5 or later), then we would see decreasedlabeling efficiency in the gonad relative to the brain, similar toobservations in the liver. However, that was not the case, sug-gesting that gonad macrophages, like microglia, are solelyprimitive in origin. The fate of fetal gonadal macrophages isunclear; however, a significant subset of macrophages in multipleadult organs is yolk-sac–derived (20). Our data show that testismacrophages are present throughout fetal development, at leastuntil birth, and may persist until adulthood in the testis, wherethey could play roles such as promoting the development of adultLeydig cells (23).Although the brain microenvironment imparts some unique

properties to microglia, their phenotype likely is attributable in

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Fig. 6. Cx3cr1-Cre is specific to fetal macrophages and can be used effec-tively for targeted macrophage ablation. (A–H) Immunofluorescence imagesof E10.5 brain (A), E10.5 yolk sac (B), E10.5 UGR (C and D), E12.5 XY gonads(E and F), and E13.5 XY gonads (G and H). C′–F′ are higher-magnificationimages of the boxed regions in C–F, respectively. Cx3cr1-Cre drives the ex-pression of Rosa-fGFP in brain microglia (A), yolk-sac macrophages (B), andUGR macrophages (C) but not in control Cre− littermates (D). Arrowheadsin A and B indicate rare GFP− F4/80+ cells. (E) Independent verification ofCx3cr1-Cre by a Rosa-Tomato reporter also shows effective Cre activationspecifically in E12.5 gonadal macrophages. (F) Tomato is not detected inSOX9+ Sertoli cells or in PECAM1+ germ cells and vasculature. (G and H)Maximum-intensity projection images of 10 optical sections equally spacedthrough the entire Z-plane of the testis show that Rosa-DTA driven byCx3cr1-Cre results in near-complete ablation of gonadal macrophages. (I)Quantification of total F4/80+ cells in a 375 × 375 × 50 μm region of E13.5control (pooled wild-type, +; Rosa-DTA, and Cx3cr1-Cre; + genotypes, n = 5)and macrophage-depleted Cx3cr1-Cre;Rosa-DTA fetal testes (n = 4). Data arerepresented as means ± SEM. (Scale bars: 50 μm.)

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part to their yolk-sac origins. Similar to microglia, which areinvolved in anastomosis of blood vessels in the brain (36), go-nadal macrophages are involved in vascular remodeling. It ispossible that the primitive hematopoietic cells in the yolk sac arepredisposed to take part in blood vessel formation and patterningand may have other unique roles during organ formation, as well.

Roles for Macrophages in Fetal Organogenesis. Growing evidencesuggests that macrophages have critical functions in tissue de-velopment apart from their classic phagocytic functions, in par-ticular in promoting angiogenesis and vascular remodeling inpostnatal organs (9). A well-characterized example is the re-finement of the hyaloid vasculature in the mouse postnatal eye,in which WNT ligands secreted by macrophages are critical forthe breakdown, remodeling, and patterning of blood vessels (52).In contrast to postnatal contexts and disease states, there are

few documented examples of the role of macrophages in fetalorganogenesis. In the fetal brain, macrophages are involved inanastomosis during vascular remodeling, because they associatewith endothelial tip cells to promote the fusion of adjacentvascular branches (36). One previous study reported that late-stage E18.5 colony stimulating factor 1 (Csf1)-deficient fetuses,which showed reduced numbers of macrophages, had decreasedinsulin cell mass and abnormal pancreatic islet morphogenesis(12). However, whether fetal macrophages affect vascularization ofthe pancreas or initial pancreatic islet differentiation was not shown.In this report, we demonstrated previously unidentified, es-

sential roles for macrophages in fetal organogenesis, using aneffective macrophage-specific cell ablation model: a Cre-re-sponsive DTA line driven by a macrophage-specific Cx3cr1-Cre.

Although previous reports of macrophage-depleted models suchas Csf1 mutants found a modest number of macrophagesremaining in fetal organs, often rescued by maternal circulatingCSF1 (53), our model specifically and effectively targets mac-rophages in a cell-autonomous manner, avoiding the confound-ing effects in earlier mutant studies as well as potential secondarytoxic side effects of diphtheria toxin administration in the Cre-inducible diphtheria toxin receptor system (54).Macrophages are known to be involved in the clearance of

apoptotic bodies and cells in development (55). Such cytotoxicactivities usually are associated with M1 macrophages. However,M2 macrophages can maintain high phagocytic activity in certaincontexts (56). Our results revealed that germ cells that fail tomigrate into the gonad but remain in the mesonephros at E11.5–E12.5 nearly always are surrounded and engulfed by macro-phages. Because germ cells can give rise to teratomas or othertumors in an inappropriate environment, they may be a priorityfor clearance by macrophages. The unusual morphology andclustering of these germ cells may reflect the initiation of anapoptotic program, part of which induces phagocytic activity ofmacrophages. In addition, Sertoli cells almost never are observedoutside the testis cords after E12.5 because of the activity ofmacrophages that normally act to refine testis cord structures bypruning mislocated Sertoli cells. Accordingly, in macrophage-ablated testes, clusters of Sertoli cells were readily found outsideof testis cords. Although our data point to a primary function ofmacrophages in vascular remodeling during testis organogen-esis, future studies are required to determine definitively ifmacrophages play critical, vascular-independent roles in tissueremodeling of the testis, i.e., if they can influence the behavior of

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Fig. 7. Macrophages are required for testis-specific vascularization and morphogenesis. Immunofluorescence images of E13.5 control +;Rosa-DTA (A, C,E, G, and I), E13.5 macrophage-depleted Cx3cr1-Cre;Rosa-DTA (B, D, F, H, and J), E12.5 control (K, M, and O), and E12.5 macrophage-depleted (L, N, and P)testes. C, D, G, and H are maximum-intensity projection images of 10 optical sections spaced equally through the entire Z-plane of the testes shown in A, B, E,and F, respectively; A, B, E, F, and I–P are single confocal optical sections. Although control testes had a robust, coherent surface coelomic artery (A and C),macrophage-depleted testes exhibited an unorganized surface vasculature (B and D). Macrophage-depleted testes also showed more vasculature remainingin the mesonephros near the gonad border (E–H) and abnormal testis cords (marked by AMH) (I and J) relative to control littermate testes. Inset in J isa higher-magnification image of the boxed region showing an aberrant Sertoli cell cluster outside the testis cord boundaries (dashed lines in Inset). E12.5control (K, M, and O) and E12.5 macrophage-depleted (L, N, and P) testes show similar levels of MKI67 (K and L), pHH3 (M and N), and Caspase-3 (O and P)expression, in particular within PECAM1+ endothelial cells (arrowheads in K–N). (Thin scale bars: 50 μm; thick scale bars: 12.5 μm.)

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Sertoli cells and/or interstitial cells directly to promote testiscord morphogenesis.Although Sertoli cells traditionally have been considered the

key cell type driving the formation of testis cord structures, theyare not sufficient for testis cord morphogenesis. The vasculatureplays an instructive role in testis morphogenesis (5–8, 39). Thefirst step of this process, the breakdown of the mesonephricvascular plexus, is dependent on gonadal–mesonephric macro-phages. Although the signals that drive vessel remodeling andtestis neovascularization still are not well-defined, Vegf signalingis required, and several lines of investigation implicated Sertolicells and/or interstitial cells as the source of VEGFA (5, 7). Inother contexts, such as in tumor vascularization, macrophageshave been implicated as a source of VEGFA production (11). Inthis study, we showed that macrophages in the fetal gonad alsoexpress Vegfa and, like tumor-associated macrophages (36–38),express the angiogenic-associated receptors NRP1 and TEK/TIE2. Macrophages may use these cell-surface receptors to se-quester pools of the endothelial attractant factors VEGF orANGPT and present them to endothelial cells to guide neo-vascularization of tumors. Consistent with this idea, VEGFA issequestered inside gonadal macrophages, perhaps influencingthe activity, behavior, or migration of endothelial cells duringtestis formation. However, our data in this study show that go-nadal macrophages are not a major source of Vegfa mRNA,suggesting that other interstitial cells produce the majority ofVEGFA required for endothelial cell proliferation and survival.Our results showing that overall endothelial cell number is notaffected in macrophage-depleted gonads are consistent with thisidea. One possibility is that macrophage-derived VEGFA is a localsource of VEGFA that influences the migratory path of endothelial cellsduring fetal testis vascular remodeling.Our data indicate that macrophages play an important role during

de novo morphogenesis of testis cords by controlling vascularizationand tissue pruning. This unexpected role for fetal macrophages maybe a developmental mechanism that is used commonly during neo-vascularization and morphogenesis of many fetal organs.

Materials and MethodsMice. CD-1 mice (Charles River) were used for wild-type expression studies.Cx3cr1-GFP mice (Cx3cr1tm1Litt) (24) are publicly available at Jackson Laboratoriesand are maintained on a mixed background of C57BL/6J (B6) and CD-1. Kdr-mCherry (also called “Flk1::myr-mCherry”) (42), maintained on a CD-1 back-ground, were a gift from M. Dickinson (Department of Molecular Physiologyand Biophysics, Baylor College of Medicine). Csf1r-CreER [Tg(Csf1r-cre/Esr1*)1Jwp](31) is a tamoxifen-inducible macrophage Cre line maintained on a mixedFVB/CD-1 background. Rosa-Tomato [Gt(ROSA)26Sortm14(CAG-tdTomato)Hze] (32),available from Jackson Laboratories, is maintained on a B6 background.Cx3cr1-Cre (also called “MW126-Cre”), a constitutive macrophage/microglialBAC-Cre line generated by the GENSAT project (57), was a gift from M. D.Gunn (Departments of Immunology and Medicine, Duke University MedicalCenter) and is maintained on a 129/B6 mixed background. Rosa-eGFP-DTA mice [Gt(ROSA)26SORtm1(DTA)Jpmb] (45) are maintained on a B6 back-ground and are publicly available from Jackson Laboratories. Farnesylated-GFPreporter mice (Rosa26R-CAG-fGFP) (46) were a gift from F. Wang (Departmentof Cell Biology, Duke University Medical Center) and are maintained ona B6 background. Tie2-Cre (also called “Tek-Cre”) [Tg(Tek-cre)1Ywa] mice(40) were a gift from R. Lang (Divisions of Pediatric Ophthalmology andDevelopmental Biology, Cincinnati Children’s Hospital Medical Center)but are publicly available from Jackson Laboratories. Mice were housedin accordance with National Institutes of Health guidelines, and experi-mental protocols were approved by the Institutional Animal Care and UseCommittee of Duke University Medical Center and/or Cincinnati Children’sHospital Medical Center.

Yolk-Sac Macrophage Lineage Tracing. To label yolk-sac progenitors specifically,we followed a previously described protocol using Csf1r-CreER (19), except thatwe used a Rosa-Tomato Cre–responsive lineage tracer (32). Csf1r-CreER maleswere crossed to Rosa-Tomato females, and pregnant females were injectedi.p. at E7.5 with 75 μg/g 4-hydroxytamoxifen (Sigma-Aldrich #H6278) and s.c.with 75 μg/g progesterone (Sigma-Aldrich #P0130). Pregnant females were

euthanized at the required stage of pregnancy, and embryos were processedfor immunofluorescence or flow cytometry as described below.

Immunofluorescence. Whole-mount immunofluorescence was performed onwhole embryos, fetal gonads and yolk sacs at E13.5 and younger as previouslydescribed (8), and on cryosections of brains, livers, and adult testes as pre-viously reported (58). Primary antibodies used for immunofluorescence arelisted in Table S1. Alexa-488– and Alexa-647–conjugated secondary anti-bodies (Molecular Probes/Life Technologies) and Dy-Lite 488 donkey anti-chicken and Cy3 donkey or goat anti-rabbit/rat secondary antibodies (Jack-son ImmunoResearch) were used at 1:500. Nuclei were stained with DAPI(Sigma) or Hoechst 33342 (Molecular Probes/Life Technologies), but nuclearstains are shown as “DAPI” in all figure panels. Samples were imaged ona Leica SP2 confocal microscope or Nikon EclipseTE2000-E microscopeequipped with an OptiGrid structured illumination imaging system.

Flow Cytometry Analysis. Fetal gonads, livers, and brains were disassociatedby 10-min, 2-min, and 3-min incubations, respectively, in 0.25% Trypsin-EDTA(Gibco/Life Technologies #2520056). Livers and brains were ruptured mechan-ically before trypsin treatment. After several washes in PBS, tissues were resus-pended in 800 μL PBS plus 4 μL DNase I (Promega #M6101). Gonads werepipetted vigorously to disrupt tissue and were passed several times througha 27-gauge needle (BD Biosciences #309623). Cell suspensions were fil-tered through a cell strainer cap (Falcon/Corning Life Sciences #352235).

After a 10-min incubation in anti-CD16/32 (FCgammaIII/II, clone 2.4G2, a giftfrom S. Thornton, Division of Rheumatology, Cincinnati Children’s HospitalMedical Center), antibody mixes were added and incubated for 20 min.Antibodies and reagents used are listed in Table S2. Cells were washed, andflow cytometry was performed on either a BD Biosciences LSRII flow cytometeror a BD Biosciences Fortessa flow cytometer. All data were analyzed usingFACS Diva software (BD Biosciences). Statistical analyses were performed viaa two-tailed Student t test.

FACS Purification of Fetal Macrophages. Macrophages were purified fromCx3cr1-GFP heterozygous embryos obtained from crossing Cx3cr1-GFP het-erozygous males to CD-1 females. GFP expression in embryos was determinedby fluorescence microscopy prior to dissection. Cells were disassociated andprepared for FACS as previously described (33). Cell purification was per-formed on a Beckman Coulter MoFlo XDP cell sorter. Cells were collected intoPBS in a 1.5-mL microcentrifuge tube, pelleted by a 5-min spin at 3,500 × g at4 °C, and stored at −80 °C until RNA extraction was performed.

PCR. RNA extraction, cDNA synthesis, and qPCR were performed as previouslydescribed (58), except that Fast SYBR Green Master Mix (Applied Biosystems/Life Technologies #4385610) was used. Expression levels were normalized toGapdh. Primers used for qPCR analysis are listed in Table S3.

Vascular-Depletion Gonad Cultures. Whole-gonad droplet cultures were per-formed on wild-type CD-1 E11.5 or E12.5 testes as previously described (41),except cultures were performed for 42–44 h. One gonad of each pair wascultured in a 30-μL droplet of control medium (DMEM/FBS/antibiotic mix-ture); the contralateral gonad was cultured in a 30-μL droplet of mediumcontaining 1.5 μg/mL VEGF receptor Tyrosine Kinase Inhibitor II (VEGFR TKIII; Calbiochem/EMD Millipore #676481–5MG). This concentration was suffi-cient to deplete vasculature effectively but did not result in increased apo-ptosis in nonendothelial cells or significantly affect Sertoli/germ cell numbersor gene expression. VEGFR TKI II targets both KDR (VEGFR2) and FLT-1(VEGFR1). Sex of embryos was confirmed by a PCR-based method (59). Threeindependent experiments were performed for each time point (E11.5 orE12.5), and within each experiment multiple XY gonads (n = 3–6) were re-moved from the mesonephros and pooled for RNA extraction. Statisticalanalyses were done using a two-tailed Student t test.

ACKNOWLEDGMENTS. We thank M. D. Gunn, F. Wang, M. Dickinson,R. Lang, and B. Hogan for mice; S. K. Dey, S. Divanovic, S. Thornton, andC. Kontos for antibodies and reagents; M. Cappelletti, M. DeLay and theCincinnati Children’s Hospital Medical Center (CCHMC) Research FlowCytometry Core for help with flow cytometry experiments; S. Potter for helpwith Rosa-DTA samples; and J. Cool and M. Czerwinski for early experimentsfocusing on macrophages. This work was funded by National Institutes of Health(NIH), Eunice Kennedy Shriver National Institute of Child Health and HumanDevelopment Grant 5R01-HD039963 and March of Dimes Grant 1-FY10-355 (toB.C.). T.D. was supported by NIH National Research Service Award PostdoctoralFellowship F32-HD058433, CCHMC developmental funds, and a CCHMC ResearchInnovation and Pilot Funding grant.

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Page 10: Yolk-sac derived macrophages regulate fetal testis ... · Yolk-sac–derived macrophages regulate fetal testis vascularization and morphogenesis Tony DeFalcoa,1, Indrashis Bhattacharyaa,

1. Brennan J, Capel B (2004) One tissue, two fates: Molecular genetic events that un-derlie testis versus ovary development. Nat Rev Genet 5(7):509–521.

2. Cool J, DeFalco T, Capel B (2012) Testis formation in the fetal mouse: Dynamic andcomplex de novo tubulogenesis. Wiley Interdiscip Rev Dev Biol 1(6):847–859.

3. Albrecht KH, Eicher EM (2001) Evidence that Sry is expressed in pre-Sertoli cells andSertoli and granulosa cells have a common precursor. Dev Biol 240(1):92–107.

4. Sekido R, Bar I, Narváez V, Penny G, Lovell-Badge R (2004) SOX9 is up-regulated by thetransient expression of SRY specifically in Sertoli cell precursors. Dev Biol 274(2):271–279.

5. Bott RC, McFee RM, Clopton DT, Toombs C, Cupp AS (2006) Vascular endothelialgrowth factor and kinase domain region receptor are involved in both seminiferouscord formation and vascular development during testis morphogenesis in the rat. BiolReprod 75(1):56–67.

6. Combes AN, et al. (2009) Endothelial cell migration directs testis cord formation. DevBiol 326(1):112–120.

7. Cool J, DeFalco TJ, Capel B (2011) Vascular-mesenchymal cross-talk through Vegf andPdgf drives organ patterning. Proc Natl Acad Sci USA 108(1):167–172.

8. DeFalco T, Takahashi S, Capel B (2011) Two distinct origins for Leydig cell progenitorsin the fetal testis. Dev Biol 352(1):14–26.

9. Pollard JW (2009) Trophic macrophages in development and disease. Nat Rev Im-munol 9(4):259–270.

10. Wynn TA, Chawla A, Pollard JW (2013) Macrophage biology in development, ho-meostasis and disease. Nature 496(7446):445–455.

11. Nucera S, Biziato D, De Palma M (2011) The interplay between macrophages andangiogenesis in development, tissue injury and regeneration. Int J Dev Biol 55(4-5):495–503.

12. Banaei-Bouchareb L, et al. (2004) Insulin cell mass is altered in Csf1op/Csf1opmacrophage-deficient mice. J Leukoc Biol 76(2):359–367.

13. Gouon-Evans V, Rothenberg ME, Pollard JW (2000) Postnatal mammary gland de-velopment requires macrophages and eosinophils. Development 127(11):2269–2282.

14. Lang RA, Bishop JM (1993) Macrophages are required for cell death and tissue re-modeling in the developing mouse eye. Cell 74(3):453–462.

15. Wiktor-Jedrzejczak WW, Ahmed A, Szczylik C, Skelly RR (1982) Hematological char-acterization of congenital osteopetrosis in op/op mouse. Possible mechanism forabnormal macrophage differentiation. J Exp Med 156(5):1516–1527.

16. Orkin SH, Zon LI (2008) Hematopoiesis: An evolving paradigm for stem cell biology.Cell 132(4):631–644.

17. Ginhoux F, et al. (2010) Fate mapping analysis reveals that adult microglia derive fromprimitive macrophages. Science 330(6005):841–845.

18. Samokhvalov IM, Samokhvalova NI, Nishikawa S (2007) Cell tracing shows the con-tribution of the yolk sac to adult haematopoiesis. Nature 446(7139):1056–1061.

19. Schulz C, et al. (2012) A lineage of myeloid cells independent of Myb and hemato-poietic stem cells. Science 336(6077):86–90.

20. Hume DA, Halpin D, Charlton H, Gordon S (1984) The mononuclear phagocyte systemof the mouse defined by immunohistochemical localization of antigen F4/80: Mac-rophages of endocrine organs. Proc Natl Acad Sci USA 81(13):4174–4177.

21. Cohen PE, Hardy MP, Pollard JW (1997) Colony-stimulating factor-1 plays a major rolein the development of reproductive function in male mice. Mol Endocrinol 11(11):1636–1650.

22. Gaytan F, Bellido C, Aguilar E, van Rooijen N (1994) Requirement for testicularmacrophages in Leydig cell proliferation and differentiation during prepubertal de-velopment in rats. J Reprod Fertil 102(2):393–399.

23. Austyn JM, Gordon S (1981) F4/80, a monoclonal antibody directed specifically againstthe mouse macrophage. Eur J Immunol 11(10):805–815.

24. Jung S, et al. (2000) Analysis of fractalkine receptor CX(3)CR1 function by targeteddeletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20(11):4106–4114.

25. Springer T, Galfré G, Secher DS, Milstein C (1979) Mac-1: A macrophage differentia-tion antigen identified by monoclonal antibody. Eur J Immunol 9(4):301–306.

26. Dai XM, et al. (2002) Targeted disruption of the mouse colony-stimulating factor 1receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increasedprimitive progenitor cell frequencies, and reproductive defects. Blood 99(1):111–120.

27. Imai Y, Ibata I, Ito D, Ohsawa K, Kohsaka S (1996) A novel gene iba1 in the majorhistocompatibility complex class III region encoding an EF hand protein expressed ina monocytic lineage. Biochem Biophys Res Commun 224(3):855–862.

28. Köhler C (2007) Allograft inflammatory factor-1/Ionized calcium-binding adaptermolecule 1 is specifically expressed by most subpopulations of macrophages andspermatids in testis. Cell Tissue Res 330(2):291–302.

29. Fleming TJ, Fleming ML, Malek TR (1993) Selective expression of Ly-6G on myeloidlineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiationantigen (Gr-1) detects members of the Ly-6 family. J Immunol 151(5):2399–2408.

30. Lipscomb MF, Masten BJ (2002) Dendritic cells: Immune regulators in health anddisease. Physiol Rev 82(1):97–130.

31. Qian BZ, et al. (2011) CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature 475(7355):222–225.

32. Madisen L, et al. (2010) A robust and high-throughput Cre reporting and character-ization system for the whole mouse brain. Nat Neurosci 13(1):133–140.

33. Jameson SA, et al. (2012) Temporal transcriptional profiling of somatic and germ cellsreveals biased lineage priming of sexual fate in the fetal mouse gonad. PLoS Genet8(3):e1002575.

34. Sica A, Mantovani A (2012) Macrophage plasticity and polarization: In vivo veritas.J Clin Invest 122(3):787–795.

35. Cao S, et al. (2002) Differential regulation of IL-12 and IL-10 gene expression inmacrophages by the basic leucine zipper transcription factor c-Maf fibrosarcoma.J Immunol 169(10):5715–5725.

36. Fantin A, et al. (2010) Tissue macrophages act as cellular chaperones for vascularanastomosis downstream of VEGF-mediated endothelial tip cell induction. Blood116(5):829–840.

37. De Palma M, et al. (2005) Tie2 identifies a hematopoietic lineage of proangiogenicmonocytes required for tumor vessel formation and a mesenchymal population ofpericyte progenitors. Cancer Cell 8(3):211–226.

38. Pucci F, et al. (2009) A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood “resident” monocytes, and embryonic macrophagessuggests common functions and developmental relationships. Blood 114(4):901–914.

39. Coveney D, Cool J, Oliver T, Capel B (2008) Four-dimensional analysis of vasculariza-tion during primary development of an organ, the gonad. Proc Natl Acad Sci USA105(20):7212–7217.

40. Kisanuki YY, et al. (2001) Tie2-Cre transgenic mice: A new model for endothelial cell-lineage analysis in vivo. Dev Biol 230(2):230–242.

41. Maatouk DM, et al. (2008) Stabilization of beta-catenin in XY gonads causes male-to-female sex-reversal. Hum Mol Genet 17(19):2949–2955.

42. Larina IV, et al. (2009) A membrane associated mCherry fluorescent reporter line forstudying vascular remodeling and cardiac function during murine embryonic de-velopment. Anat Rec (Hoboken) 292(3):333–341.

43. Holness CL, da Silva RP, Fawcett J, Gordon S, Simmons DL (1993) Macrosialin, a mousemacrophage-restricted glycoprotein, is a member of the lamp/lgp family. J Biol Chem268(13):9661–9666.

44. Rabinowitz S, Horstmann H, Gordon S, Griffiths G (1992) Immunocytochemical char-acterization of the endocytic and phagolysosomal compartments in peritoneal mac-rophages. J Cell Biol 116(1):95–112.

45. Ivanova A, et al. (2005) In vivo genetic ablation by Cre-mediated expression ofdiphtheria toxin fragment A. Genesis 43(3):129–135.

46. Rawlins EL, et al. (2009) The role of Scgb1a1+ Clara cells in the long-term mainte-nance and repair of lung airway, but not alveolar, epithelium. Cell Stem Cell 4(6):525–534.

47. Hutson JC (1990) Changes in the concentration and size of testicular macrophagesduring development. Biol Reprod 43(5):885–890.

48. Livera G, Delbes G, Pairault C, Rouiller-Fabre V, Habert R (2006) Organotypic culture,a powerful model for studying rat and mouse fetal testis development. Cell Tissue Res324(3):507–521.

49. Breucker H (1978) Macrophages, a normal component in seasonally involuting testesof the swan, Cygnus olor. Cell Tissue Res 193(3):463–471.

50. Dubey N, Lal B (2009) Paracrine role of macrophage produced-nitric oxide (NO) inLeydig cell steroidogenesis in a teleost, Clarias batrachus: Impact of gonadotropin,growth hormone and insulin on NO production by testicular macrophages. Gen CompEndocrinol 160(1):12–18.

51. Khan UW, Rai U (2007) Differential effects of histamine on Leydig cell and testicularmacrophage activities in wall lizards: Precise role of H1/H2 receptor subtypes.J Endocrinol 194(2):441–448.

52. Lobov IB, et al. (2005) WNT7b mediates macrophage-induced programmed cell deathin patterning of the vasculature. Nature 437(7057):417–421.

53. Roth P, Dominguez MG, Stanley ER (1998) The effects of colony-stimulating factor-1on the distribution of mononuclear phagocytes in the developing osteopetroticmouse. Blood 91(10):3773–3783.

54. Buch T, et al. (2005) A Cre-inducible diphtheria toxin receptor mediates cell lineageablation after toxin administration. Nat Methods 2(6):419–426.

55. Henson PM, Hume DA (2006) Apoptotic cell removal in development and tissue ho-meostasis. Trends Immunol 27(5):244–250.

56. Leidi M, et al. (2009) M2 macrophages phagocytose rituximab-opsonized leukemictargets more efficiently than m1 cells in vitro. J Immunol 182(7):4415–4422.

57. Gong S, et al. (2003) A gene expression atlas of the central nervous system based onbacterial artificial chromosomes. Nature 425(6961):917–925.

58. Defalco T, Saraswathula A, Briot A, Iruela-Arispe ML, Capel B (2013) Testosteronelevels influence mouse fetal Leydig cell progenitors through notch signaling. BiolReprod 88(4):91.

59. Clapcote SJ, Roder JC (2005) Simplex PCR assay for sex determination in mice. Bio-techniques 38(5):702, 704, 706.

DeFalco et al. PNAS | Published online May 27, 2014 | E2393

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