ABSTRACT

The discovery of a fetal origin for tissue-resident macrophages (trMacs) has inspired an intense search for the mechanisms underlying their development. Here, we performed in vivo lineage tracing of cells with an expression history of IL7Rα, a marker exclusively associated with the lymphoid lineage in adult hematopoiesis. Surprisingly, we found that Il7r-Cre labeled fetal-derived, adult trMacs. Labeling was almost complete in some tissues and partial in others. The putative progenitors of trMacs, yolk sac (YS) erythromyeloid progenitors, did not express IL7R, and YS hematopoiesis was unperturbed in IL7R-deficient mice. In contrast, tracking of IL7Rα message levels, surface expression, and Il7r-Cre-mediated labeling across fetal development revealed dynamic regulation of Il7r mRNA expression and rapid upregulation of IL7Rα surface protein upon transition from monocyte to macrophage within fetal tissues. Fetal monocyte differentiation in vitro produced IL7R+ macrophages, supporting a direct progenitor-progeny relationship. Additionally, blockade of IL7R function during late gestation specifically impaired the establishment of fetal-derived trMacs in vivo. These data provide evidence for a distinct function of IL7Rα in fetal myelopoiesis and identify IL7R as a novel regulator of trMac development.

INTRODUCTION

Hematopoietic stem cells (HSCs) are responsible for sustaining blood and immune cell production across the lifespan of the animal, under steady-state conditions, during infection, and following transplantation. However, recent findings have revealed that many tissue-resident immune cells are poorly generated or regenerated from adult HSCs, including subsets of tissue-resident macrophages (trMacs), such as microglia, epidermal Langerhans cells, liver Kupffer cells, and alveolar macrophages (Beaudin and Forsberg, 2016; Cool and Forsberg, 2019; Ginhoux et al., 2010; Gomez Perdiguero et al., 2015; Guilliams et al., 2013; Hashimoto et al., 2013; Hoeffel et al., 2015, 2012; Sawai et al., 2016; Yona et al., 2013). These trMacs are specialized macrophages that reside within tissues and have specific functions in tissue and immune homeostasis (Epelman et al., 2014; Ginhoux et al., 2010; Gomez Perdiguero et al., 2015; Guilliams et al., 2013; Hoeffel et al., 2012). Unlike classical adult monocyte-derived macrophages, which are recruited from circulation upon inflammation and have high turnover rates, trMacs are locally maintained by proliferation, independently of adult hematopoiesis (Ajami et al., 2007; Hashimoto et al., 2013; Hulsmans et al., 2017; Merad et al., 2002). Recent fate-mapping studies have revealed a fetal origin of specific trMacs (Epelman et al., 2014; Gomez Perdiguero et al., 2015; Hoeffel et al., 2015; Schulz et al., 2012; Yona et al., 2013), but the cellular and molecular mechanisms driving trMac establishment and expansion within fetal tissues are poorly understood.

Whether trMacs are seeded once from a common progenitor or in reiterative waves throughout development is under intense investigation. Accumulating evidence points towards extra-embryonic yolk sac (YS)-derived erythromyeloid progenitors (EMPs) as the initial cell of origin for macrophages that seed developing tissues, and the primary source of brain microglia (Gomez Perdiguero et al., 2015; Kierdorf et al., 2013). Microglia and other trMacs can differentiate directly from EMPs through either Myb-dependent or -independent pathways, likely through an EMP-derived intermediate (Hoeffel et al., 2015; Schulz et al., 2012). Macrophages derived from fetal liver (FL) precursors may replace or replenish certain fetal-derived trMac populations initially seeded by YS-derived cells, including lung alveolar macrophages, epidermal Langerhans cells and liver Kupffer cells (Guilliams et al., 2013; Hoeffel et al., 2015, 2012; Yona et al., 2013). However, both the precise cell of origin for FL precursors and their specific contribution to adult trMac compartments remain controversial, partly because of incomplete progenitor labeling and the difficulties in accurately tracking progeny in situ. Despite intense investigation of the mechanisms regulating macrophage differentiation from YS progenitors, including gene expression programs (Mass et al., 2016) and mechanisms of tissue seeding (Stremmel et al., 2018), comparatively less is known about the developmental mechanisms that regulate the establishment of tissue macrophages from later waves of hematopoietic cell production.

Here, our investigation of hematopoietic development using the Il7r-Cre lineage-tracing model (Schlenner et al., 2010) revealed unexpected and robust labeling of adult trMacs in multiple tissues. Examination of fetal myeloid development revealed a transient wave of IL7Rα expression in developing fetal macrophages that was dynamically regulated as macrophages differentiated within developing resident tissues. Both germline deletion and specific blockade of IL7R during the developmental window in which trMacs express IL7R confirmed the functional requirement for IL7R signaling during fetal trMac development. In contrast, IL7Rα was not expressed by YS EMPs and YS hematopoiesis was unperturbed in Il7r−/− embryos. Together, these experiments reveal IL7R as a novel regulator of fetal macrophage development during late gestation.

RESULTS

Il7r-Cre specifically labels adult tissue-resident macrophages

The lymphoid-associated genes Flk2 (Flt3), Il7r, and Rag1 label cells with increasingly restricted lymphoid potential in adult hematopoiesis (Adolfsson et al., 2005; Forsberg et al., 2006; Igarashi et al., 2002; Kondo et al., 1997). In contrast, during fetal development all three markers identify oligopotent hematopoietic progenitors with both myeloid and lymphoid potential (Beaudin et al., 2016; Boiers et al., 2013). Although we and others have used the Flk2-Cre and Rag1-Cre models to track the contribution of fetal progenitors to trMac populations (Boiers et al., 2013; Epelman et al., 2014; Gomez Perdiguero et al., 2015; Hashimoto et al., 2013; Hoeffel et al., 2015), the contribution of IL7R-marked progenitors to the same populations has not been previously examined. To decipher the contribution of specific transient hematopoietic progenitors to adult trMacs, we compared adult trMac labeling across three lineage-tracing models: Flk2-Cre, Il7r-Cre and Rag1-Cre. We crossed mice expressing Rag1-Cre (Welner et al., 2009) or Il7r-Cre (Schlenner et al., 2010) to mTmG mice expressing a dual-color fluorescent reporter (Muzumdar et al., 2007), thereby creating ‘Rag1Switch’ and ‘IL7RSwitch’ models (Fig. 1A) analogous to the previously described FlkSwitch mouse (Beaudin et al., 2016; Boyer et al., 2012, 2011). In all models, all cells express Tomato (Tom) until Cre-mediated recombination results in the irreversible switch to GFP expression by that cell and all of its progeny (Fig. 1B).

Fig. 1.

Il7r-Cre specifically labels adult tissue-resident macrophage populations. Representative flow cytometric analysis of reporter expression across different monocyte and macrophage populations in adult mice. Tomato (Tom) and GFP expression is highlighted by red and green boxes, respectively, in FlkSwitch, Rag1Switch and IL7Rswitch models. Values indicate mean frequencies±s.e.m. of gated Il7r-Cre marked GFP+ populations. Plots and values are representative of four or five mice each representing three independent experiments. (A) Schematic of the ‘Switch’ models. Cre recombinase expression was controlled by either Flk2, IL7Rα or Rag1 regulatory elements, respectively. Cre-driver mice were crossed to mice expressing a dual-color reporter expressing either Tom or GFP, under control of the Rosa26 locus. Expression of Cre results in an irreversible genetic deletion event that causes a switch in reporter expression from Tom to GFP. (B) Schematic of Cre-mediated reporter switching in the ‘switch’ models. All cells initially express Tom. Expression of Cre results in an irreversible switch from Tomato to GFP expression. Once a cell expresses GFP, it can only give rise to GFP-expressing progeny. (C) Representative flow cytometric analysis of reporter expression in circulating CD11bhiGrmid monocytes in the peripheral blood of adult FlkSwitch, Rag1Switch and IL7RSwitch mice. (D) Representative flow cytometric analysis of reporter expression in LCs (F4/80+CD11b+CD11cmid) in the epidermis of adult FlkSwitch, Rag1Switch and IL7RSwitch mice. (E) Representative flow cytometric analysis of reporter expression in microglia (CD45+F4/80hiCD11bhiLy6gCD11c) in the brains of FlkSwitch, Rag1Switch and IL7RSwitch adult mice. For additional gating see Fig. S1H. (F) Representative flow cytometric analysis of reporter expression in lung AMs (CD45+F4/80hiCD11bmid SiglecF+CD11c+) of FlkSwitch, Rag1Switch, and IL7RSwitch adult mice. G, Representative flow cytometric analysis of reporter expression in liver KCs (CD45+F4/80hiCD11bmidCD169+) in adult FlkSwitch, Rag1Switch and IL7RSwitch mice. (H) Lack of IL7Rα surface expression in the LCs of the epidermis, brain microglia, lung AMs, and liver KCs. For each tissue, IL7Rα surface expression of gated population is shown for two representative mice in blue. Gray shaded area represents fluorescence-minus-one (FMO) control. (I) Quantitative RT-PCR analysis of Il7r expression in sorted bone marrow-derived B220+CD43+ Pro-B cells and CD11b+Grmid monocytes, and LCs of the epidermis, brain microglia, lung AMs, and liver KCs isolated from WT adult mice. Data represent mean±s.e.m. for three independent experiments. Values are normalized to Pro-B cells, set to 100. ND, not detected. Additional analyses of adult tissue myeloid populations can be found in Fig. S1.

Fig. 1.

Il7r-Cre specifically labels adult tissue-resident macrophage populations. Representative flow cytometric analysis of reporter expression across different monocyte and macrophage populations in adult mice. Tomato (Tom) and GFP expression is highlighted by red and green boxes, respectively, in FlkSwitch, Rag1Switch and IL7Rswitch models. Values indicate mean frequencies±s.e.m. of gated Il7r-Cre marked GFP+ populations. Plots and values are representative of four or five mice each representing three independent experiments. (A) Schematic of the ‘Switch’ models. Cre recombinase expression was controlled by either Flk2, IL7Rα or Rag1 regulatory elements, respectively. Cre-driver mice were crossed to mice expressing a dual-color reporter expressing either Tom or GFP, under control of the Rosa26 locus. Expression of Cre results in an irreversible genetic deletion event that causes a switch in reporter expression from Tom to GFP. (B) Schematic of Cre-mediated reporter switching in the ‘switch’ models. All cells initially express Tom. Expression of Cre results in an irreversible switch from Tomato to GFP expression. Once a cell expresses GFP, it can only give rise to GFP-expressing progeny. (C) Representative flow cytometric analysis of reporter expression in circulating CD11bhiGrmid monocytes in the peripheral blood of adult FlkSwitch, Rag1Switch and IL7RSwitch mice. (D) Representative flow cytometric analysis of reporter expression in LCs (F4/80+CD11b+CD11cmid) in the epidermis of adult FlkSwitch, Rag1Switch and IL7RSwitch mice. (E) Representative flow cytometric analysis of reporter expression in microglia (CD45+F4/80hiCD11bhiLy6gCD11c) in the brains of FlkSwitch, Rag1Switch and IL7RSwitch adult mice. For additional gating see Fig. S1H. (F) Representative flow cytometric analysis of reporter expression in lung AMs (CD45+F4/80hiCD11bmid SiglecF+CD11c+) of FlkSwitch, Rag1Switch, and IL7RSwitch adult mice. G, Representative flow cytometric analysis of reporter expression in liver KCs (CD45+F4/80hiCD11bmidCD169+) in adult FlkSwitch, Rag1Switch and IL7RSwitch mice. (H) Lack of IL7Rα surface expression in the LCs of the epidermis, brain microglia, lung AMs, and liver KCs. For each tissue, IL7Rα surface expression of gated population is shown for two representative mice in blue. Gray shaded area represents fluorescence-minus-one (FMO) control. (I) Quantitative RT-PCR analysis of Il7r expression in sorted bone marrow-derived B220+CD43+ Pro-B cells and CD11b+Grmid monocytes, and LCs of the epidermis, brain microglia, lung AMs, and liver KCs isolated from WT adult mice. Data represent mean±s.e.m. for three independent experiments. Values are normalized to Pro-B cells, set to 100. ND, not detected. Additional analyses of adult tissue myeloid populations can be found in Fig. S1.

We compared reporter expression in fetal-derived trMacs of all three models to reporter expression in adult bone marrow (BM)-derived circulating peripheral blood (PB) monocytes (CD11bhiGr1loSSclo; CD11b also known as ITGAM, Gr1 as Ly6G; SSc, side scatter parameter), the precursors of adult HSC-derived macrophages. As expected, Cre-driven GFP labeling of monocytes in IL7RSwitch and Rag1Switch mice was less than 5% (Fig. 1C) and paralleled the nominal labeling observed in adult HSCs and myeloid progenitors (Fig. S1A,B), as previously reported (Schlenner et al., 2010; Welner et al., 2009). Adult FlkSwitch mice exhibited high GFP labeling in PB monocytes (Fig. 1C), as we previously reported (Boyer et al., 2011). Intriguingly, examination of Cre-driven reporter switching in adult trMacs revealed distinct GFP labeling patterns across all three lineage-tracing models and across tissues. Within Langerhans cells (LCs) of the epidermis, around 20% of cells were labeled by GFP in the FlkSwitch model (Fig. 1D), consistent with previous reports (Gomez Perdiguero et al., 2015; Hoeffel et al., 2015). Low GFP labeling was observed in LCs of Rag1Switch mice (Fig. 1D), as described previously for skin and other tissue macrophages during fetal development (Boiers et al., 2013). In sharp contrast, up to 96% of LCs expressed GFP in IL7RSwitch mice (Fig. 1D). A similar pattern was observed for microglia; consistent with a pre-HSC origin; virtually no reporter switching was observed in adult microglia of FlkSwitch mice (Fig. 1E) and minimal microglia labeling was observed in Rag1Switch mice. Remarkably, however, GFP labeling of microglia was over 85% in IL7RSwitch mice (Fig. 1E).

Examination of reporter expression across lineage-tracing models in trMacs of the lung (alveolar macrophages, AMs) and the liver (Kupffer cells, KCs) yielded a different labeling pattern compared with the LCs and microglia (Fig. 1F,G). In FlkSwitch and Rag1Switch mice, GFP labeling in AMs and KCs was low and roughly comparable to LCs (∼8-19% GFP+ cells; Fig. 1D,F,G). Surprisingly, in IL7RSwitch mice, GFP labeling in AM and KC populations was substantially higher, ∼40%, compared with both FlkSwitch and Rag1Switch models (Fig. 1F,G). Although labeling in these two populations was considerably less than that of LCs or microglia in the same mice (∼85-96%), it was substantially higher than labeling of adult BM-derived myeloid populations (F4/80loCD11b+; F4/80 also known as ADGRE1) within the same tissues (<5%) (Fig. S1C-E). Comparison of Cre-mediated labeling between circulating BM-derived monocytes (<4%; Fig. 1C) and trMacs (40-90%; Fig. 1D-G) in adult IL7RSwitch mice also revealed stark differences in labeling, supporting previous reports that adult BM-derived monocytes did not substantially contribute (at steady-state) to the trMac populations investigated, including microglia, LCs, KCs, and AMs (Gomez Perdiguero et al., 2015; Hashimoto et al., 2013; Hoeffel et al., 2012; Sheng et al., 2015). Similarly, other myeloid cells in the tissues, such as neutrophils, were also minimally labeled (Fig. S1F,G), consistent with a previous report (Schlenner et al., 2010). Despite substantial Il7r-Cre-mediated labeling, IL7Rα surface expression was undetectable in any adult trMacs surveyed (Fig. 1H). Il7r was also virtually undetectable in trMacs compared with BM Pro-B-cells (Fig. 1I). The robust labeling of adult trMacs in the IL7RSwitch model in the absence of message or surface expression therefore suggested that IL7Rα was expressed during an earlier window of macrophage development.

YS hematopoiesis does not depend on IL7R

YS EMPs have been proposed as a cell of origin for fetal trMac development, either through direct differentiation or through the Myb-dependent generation of intermediate progenitors that seed the fetal liver (Gomez Perdiguero et al., 2015; Hoeffel et al., 2015, 2012; Mass et al., 2016; Schulz et al., 2012). To determine whether Il7r-Cre labeling of adult trMac populations initiated in YS EMPs during embryonic development, we examined Il7r-Cre-driven GFP labeling, IL7Rα surface expression and Il7r in YS EMPs and YS macrophages. YS EMPs (Ter119Kit+CD41+; Ter119 also known as LY67; CD41 also known as ITGA2B) at embryonic day (E)9.5 were minimally labeled by Il7r-Cre, consistent with a complete absence of IL7Rα surface expression (Fig. 2A,A′). There was virtually no Il7r-Cre-induced reporter expression within the CD45+ (CD45 also known as Ptprc) fraction of the YS, which included myeloid cells expressing CD11b or F4/80 (Fig. 2B), and there was similarly no IL7Rα surface expression observed (Fig. 2B′). Il7r in E9.5 EMPs was detectable at very high Ct values (Fig. 2C,C′), and consequently we observed slightly more labeling in YS EMPs by E10.5 (Fig. 2A). However, increased reporter expression was not accompanied by surface expression (Fig. 2A′). Overall, labeling of YS EMPs was comparable to background labeling observed in adult hematopoiesis (Schlenner et al., 2010). To clarify whether IL7R expression regulated YS hematopoiesis, we examined the frequency of YS EMPs and YS macrophages in Il7r−/− embryos, which have a germline deletion of IL7Rα (Peschon et al., 1994). Neither YS EMPs nor YS macrophage frequency was significantly different between wild-type (WT) and Il7r−/− mice (Fig. 2D,E). Together, these data indicate that IL7R is not required for the generation of YS progenitors or YS macrophages, and suggest that the robust labeling of F4/80hi trMacs by Il7r-Cre occurs later in trMac ontogeny.

Fig. 2.

IL7Rα is dispensable for YS hematopoiesis. (A,A′) Representative FACS plots showing gating strategy, Il7r-Cre-driven GFP labeling, and IL7Rα surface expression in Ter119Kit+CD41+ EMPs in the YS at E9.5, E10.5, E11.5 and in the AGM at E11.5. Red and green boxes denote gates for Tom+ and GFP+ progenitors, respectively. Gray shaded areas in histograms indicate FMO control. Plots and values indicate mean frequencies±s.e.m. of gated Il7r-Cre-marked GFP+ populations for four to seven mice from at least two independent experiments. (B) Representative FACS plots showing gating, Il7r-Cre-driven GFP labeling, and IL7Rα surface expression in CD45+Ter119 cells in the YS at E9.5. Adjacent plot shows CD11b and F4/80 expression within the CD45+Ter119 compartment. Red and green boxes denote gates for Tom and GFP expression of CD45+Ter119 cells, respectively. (C,C′) Quantitative RT-PCR analysis of Il7r mRNA in YS EMPs from E9.5. (C) ΔΔCT values normalized to beta-actin compared with Il7r expression levels in monocyte dendritic progenitors (MDPs; CD115+Kit+Flk2+Ly6c) isolated from E14.5 FL. (C′) Representative amplification plot of beta-actin (solid lines) and Il7r (dashed lines) for quantitative RT-PCR of E14.5 MDPs and EMPs as described in C. Data show mean±s.e.m. from three independent experiments. (D,E) Quantification of the frequency of EMPs (D) and F4/80+ macrophages (E) in YS of Il7r−/− and WT embryos at E9.5. Data show mean frequency±s.e.m. representing three independent experiments. (F,G) Representative FACS plots showing gating, Il7r-Cre-driven GFP labeling, and IL7Rα surface expression in cKit+LinSca1+ stem and progenitor cells at E12.5 (F) and E14.5 (G). (H) Quantification across development of the percentage (±s.e.m.) of Il7r-Cre-driven reporter labeling (GFP+) in the phenotypically defined stem and progenitor populations described in A-G. Additional analysis of Il7r-Cre-driven reporter labeling in fetal hematopoietic populations can be found in Fig. S2.

Fig. 2.

IL7Rα is dispensable for YS hematopoiesis. (A,A′) Representative FACS plots showing gating strategy, Il7r-Cre-driven GFP labeling, and IL7Rα surface expression in Ter119Kit+CD41+ EMPs in the YS at E9.5, E10.5, E11.5 and in the AGM at E11.5. Red and green boxes denote gates for Tom+ and GFP+ progenitors, respectively. Gray shaded areas in histograms indicate FMO control. Plots and values indicate mean frequencies±s.e.m. of gated Il7r-Cre-marked GFP+ populations for four to seven mice from at least two independent experiments. (B) Representative FACS plots showing gating, Il7r-Cre-driven GFP labeling, and IL7Rα surface expression in CD45+Ter119 cells in the YS at E9.5. Adjacent plot shows CD11b and F4/80 expression within the CD45+Ter119 compartment. Red and green boxes denote gates for Tom and GFP expression of CD45+Ter119 cells, respectively. (C,C′) Quantitative RT-PCR analysis of Il7r mRNA in YS EMPs from E9.5. (C) ΔΔCT values normalized to beta-actin compared with Il7r expression levels in monocyte dendritic progenitors (MDPs; CD115+Kit+Flk2+Ly6c) isolated from E14.5 FL. (C′) Representative amplification plot of beta-actin (solid lines) and Il7r (dashed lines) for quantitative RT-PCR of E14.5 MDPs and EMPs as described in C. Data show mean±s.e.m. from three independent experiments. (D,E) Quantification of the frequency of EMPs (D) and F4/80+ macrophages (E) in YS of Il7r−/− and WT embryos at E9.5. Data show mean frequency±s.e.m. representing three independent experiments. (F,G) Representative FACS plots showing gating, Il7r-Cre-driven GFP labeling, and IL7Rα surface expression in cKit+LinSca1+ stem and progenitor cells at E12.5 (F) and E14.5 (G). (H) Quantification across development of the percentage (±s.e.m.) of Il7r-Cre-driven reporter labeling (GFP+) in the phenotypically defined stem and progenitor populations described in A-G. Additional analysis of Il7r-Cre-driven reporter labeling in fetal hematopoietic populations can be found in Fig. S2.

Il7r-Cre labels non-HSC progenitors in the fetal liver

As early YS progenitors and macrophages were minimally labeled by Il7r-Cre, we investigated labeling in putative progenitors slightly later in development. Labeling remained comparably low in both YS and aorta-gonad-mesonephros (AGM) Kit+CD41+ EMPs at E11.5, and IL7R surface expression was still undetectable in the EMPs within the FL (Fig. 2A,A′; Fig. S2A). Higher Il7r-Cre-driven labeling was observed in FL Lin-Kit+Sca1+ (KLS) progenitors by E12.5 (18%), but surface expression was still low (Fig. 2F,H). Labeling subsequently declined in E14.5 progenitors (Fig. 2G,H; P<0.001), and all labeling in E14.5 progenitors was confined to the CD150 (SLAMF1)-multipotent progenitor compartment (Fig. S2B). Consistent with labeling of upstream multipotent cells in the FL, comparable Il7r-Cre labeling was observed in all downstream progenitor and mature compartments (Fig. S2C-G), with the exception of significant labeling in committed lymphoid progenitors (Fig. S2D) and mature lymphoid cells (Fig. S2E). Labeled progenitors at E14.5 did not possess long-term multilineage reconstitution, confirming that they were not definitive HSCs (Fig. S2H). These data suggest that IL7R reporter expression in late (E10.5) YS and AGM partially labels a transient progenitor that contributes to FL hematopoiesis but is not a definitive HSC.

Il7r-Cre labeling of myeloid cells is tissue and developmental stage specific

Despite low levels of labeling in YS and fetal liver progenitors, the Il7r-Cre labeling in adult trMacs (Fig. 1) was considerably higher compared with any progenitor we profiled across fetal development (Fig. 2H; Fig. S2). We reasoned that progenitor labeling alone could not entirely explain the labeling observed in those compartments. To resolve this discrepancy, we next evaluated Il7r-Cre-driven labeling and IL7Rα expression in myeloid-restricted precursors within peripheral tissues during FL stage development. We initiated our investigation at E12.5, the stage at which significant progenitor labeling by Il7r-Cre was initially observed (Fig. 2F,H). At this stage of development, macrophages derived from the extra-embryonic YS (F4/80hiCD11blo) are already present in the tissues, but may be replaced by incoming FL-derived macrophage precursors or monocytes (F4/80loCD11bmid/hi; Fig. 3A; Fig. S3) (Hoeffel et al., 2015). F4/80loCD11bmid/hi macrophage precursors in fetal peripheral tissues are heterogeneous or low for Ly6c expression and express the fetal macrophage marker CD64 (FCGR1) (Fig. S4A-C; Hoeffel et al., 2015), suggesting their propensity to differentiate into macrophages.

Fig. 3.

Il7r-Cre dynamically labels myeloid cells during tissue-resident macrophage development. (A) Representative flow cytometric analysis indicating gating for FL-derived F4/80lo monocytes (live Ter119CD45+F4/80loCD11bmid/hi; orange gates) and previously seeded F4/80hi macrophages (live Ter119CD45+F4/80hiCD11bmid; purple gates) in different tissues at E12.5, E14.5 and P0. Pre-gates are shown in Fig. S3. (B) Quantification of Il7r-Cre-driven GFP labeling within gated population indicated in A across different tissues at E12.5, E14.5, P0 and Adult (8-12 weeks). Orange asterisks denote cross-tissue differences in GFP labeling between F4/80lo monocytes and purple asterisks denote cross-tissue differences in GFP labeling between F4/80hi macrophages. Black asterisks denote differences in GFP labeling between F4/80lo monocytes and F4/80hi macrophages within each tissue. n=4-11 representing at least three independent experiments. n.a., not analyzed. (C) Time course of Il7r-Cre-driven GFP labeling in F4/80hi macrophages and F4/80lo monocytes shown in B across ontogeny. GFP labeling was quantified in adult tissue macrophages as phenotypically defined in Fig. 1. Asterisks indicate statistically significant differences between the percentage of GFP labeling at the previously measured time point, with purple asterisks denoting differences between the indicated F4/80hi macrophages and orange asterisks denoting differences between F4/80lo monocyte populations. Representative pre-gating for cell populations in Fig. 3A are shown in Fig. S3. *P<0.05, **P<0.005, ***P<0.0005. Error bars represent s.e.m.

Fig. 3.

Il7r-Cre dynamically labels myeloid cells during tissue-resident macrophage development. (A) Representative flow cytometric analysis indicating gating for FL-derived F4/80lo monocytes (live Ter119CD45+F4/80loCD11bmid/hi; orange gates) and previously seeded F4/80hi macrophages (live Ter119CD45+F4/80hiCD11bmid; purple gates) in different tissues at E12.5, E14.5 and P0. Pre-gates are shown in Fig. S3. (B) Quantification of Il7r-Cre-driven GFP labeling within gated population indicated in A across different tissues at E12.5, E14.5, P0 and Adult (8-12 weeks). Orange asterisks denote cross-tissue differences in GFP labeling between F4/80lo monocytes and purple asterisks denote cross-tissue differences in GFP labeling between F4/80hi macrophages. Black asterisks denote differences in GFP labeling between F4/80lo monocytes and F4/80hi macrophages within each tissue. n=4-11 representing at least three independent experiments. n.a., not analyzed. (C) Time course of Il7r-Cre-driven GFP labeling in F4/80hi macrophages and F4/80lo monocytes shown in B across ontogeny. GFP labeling was quantified in adult tissue macrophages as phenotypically defined in Fig. 1. Asterisks indicate statistically significant differences between the percentage of GFP labeling at the previously measured time point, with purple asterisks denoting differences between the indicated F4/80hi macrophages and orange asterisks denoting differences between F4/80lo monocyte populations. Representative pre-gating for cell populations in Fig. 3A are shown in Fig. S3. *P<0.05, **P<0.005, ***P<0.0005. Error bars represent s.e.m.

Analysis of Il7r-Cre-driven reporter expression revealed two very clear patterns: first, GFP labeling at E12.5 was significantly higher in all F4/80lo monocytes compared with F4/80hi macrophages across all tissues (Fig. 3B, E12.5). Second, cross-tissue comparison revealed higher GFP labeling in F4/80lo monocytes in brain, lung and epidermis compared with liver (Fig. 3B, E12.5). Across all F4/80hi macrophages, GFP labeling was highest in the skin (25%; P<0.05 compared with liver F4/80hi macrophages). A very comparable pattern emerged at E14.5; however, between E12.5 and E14.5, GFP labeling increased slightly across all F4/80hi macrophage populations, but only significantly for the skin (Fig. 3C, Skin/Epi).

From late gestation into early neonatal development, the patterns of Il7r-Cre-mediated GFP labeling among F4/80hi macrophages and F4/80lo monocytes were strikingly different, both between cell types and between tissues (Fig. 3B,C). Beyond E17.5, GFP labeling in liver and lung was higher in F4/80hi macrophages compared with F4/80lo monocytes. This ‘switch’ reflected a significant increase in GFP labeling of F4/80hi macrophages between E14.5 and postnatal day (P)0 and a concomitant decrease in F4/80lo monocyte labeling (Fig. 3C), coincident with the HSC-dependent contribution to fetal monocytes later in gestation (Hoeffel et al., 2015; Yona et al., 2013). GFP labeling in lung and liver macrophages reached adult levels by P0 (Fig. 3B, Fig. 1F,G; P>0.5). The increase in labeling of F4/80hi macrophages across fetal development suggested either that Il7r-Cre labeling was occurring cell-autonomously in F4/80hi macrophages, or that Il7r-labeled fetal monocytes were gradually replacing or contributing to tissue macrophage populations in the lung and liver during fetal hematopoiesis.

In contrast to lung and liver, progression of GFP labeling within the skin and brain across ontogeny relayed a different pattern. F4/80lo monocytes were barely detectable in the epidermis and brain after E14.5 (Fig. 3A, bottom right panels), as previously described (Hoeffel et al., 2015). GFP labeling of skin macrophages increased robustly from E17.5 to P0 (Fig. 3C, Skin/Epi), and continued to increase postnatally (Fig. 3B,C). Similarly, microglia labeling increased steadily across development, but had still not reached adult levels by P14 (Fig. 3B,C). The disparate GFP labeling that we observed across different tissues suggested a differential involvement of IL7R in the development of different trMac populations across ontogeny.

IL7Rα expression is dynamically regulated during fetal tissue macrophage development

To gain insight into what was driving the observed pattern of Il7r-Cre-mediated GFP labeling within F4/80lo monocyte and F4/80hi macrophage populations across development, we probed Il7r levels beginning at E14.5 (Fig. 4A). Across tissues, F4/80lo monocytes expressed low but consistently detectable levels of Il7r relative to fetal liver monocyte dendritic progenitors (MDPs), which are known to express Il7r (Hoeffel et al., 2015; Fig. 4A). Circulating peripheral blood Ly6chi monocytes also expressed detectable Il7r and were labeled by Il7r-Cre to a similar degree as liver F4/80lo monocytes (Fig. S4C,D). Increased Il7r mRNA in peripheral tissue F4/80lo monocytes corresponded with higher Cre-mediated GFP expression (Fig. 4A), as GFP labeling increased in monocytes once they had exited the liver at E14.5 (Fig. 3B). Lung and skin monocytes with the highest GFP labeling also expressed the highest levels of Il7r mRNA. In comparison, and consistent with significantly lower Cre-mediated GFP labeling, Il7r was virtually undetectable in F4/80hi macrophages (Fig. 4A) at E14.5. Postnatally, at P14, Il7r levels continued to be negligible in macrophages of the lung and liver, but microglia and LCs expressed detectable Il7r (Fig. 4B), driving continued postnatal Cre-mediated recombination in those tissues. These data confirmed the fidelity of the Il7r-Cre model and revealed Il7r expression by fetal myeloid cells during development.

Fig. 4.

IL7Rα message and surface expression are dynamically regulated during macrophage development. (A) Quantitative RT-PCR analysis of Il7r mRNA in monocytes (F4/80loCD11bhi) and macrophages (F4/80hiCD11blo) isolated from liver, lung, brain and skin of E14.5 fetuses. Fetal liver macrophage dendritic cell precursors (MDP; CD115+Kit+Flk2–Ly6cCD11b) and adult BM monocytes (CD11b+Gr1mid) were used as positive and negative controls, respectively. Data shown are mean±s.e.m. of ΔΔCT values calculated for Il7r and β-actin and normalized to MDPs (set to 100) across three independent experiments. (B) Quantitative RT-PCR analysis of Il7r mRNA in monocytes (F4/80loCD11bhi) and macrophages (F4/80hiCD11blo) isolated from liver, lung, brain and epidermis (Epi) of neonatal mice at P14. Adult BM Pro-B cells (B220+CD43+) and BM monocytes (CD11b+Gr1+) were used as positive and negative controls, respectively. Data shown are mean±s.e.m. of ΔΔCT values calculated for Il7r and beta-actin and normalized to BM Pro-B cells (set to 100) across three independent experiments. ND, not detected. (C,D) Representative flow cytometric analysis of IL7Rα surface expression in F4/80lo monocytes (CD45+F4/80loCD11bhi; C) and F4/80hi macrophages (CD45+F4/80hiCD11bmid; D) in different tissues at E12.5, E14.5, E17.5, P0 and P14. Gray shaded area indicates FMO control shown for each cell type, respectively. Plots are representative of analysis in five or six mice from three independent experiments. Analysis of common gamma chain expression in fetal tissue macrophages can be found in Fig. S4.

Fig. 4.

IL7Rα message and surface expression are dynamically regulated during macrophage development. (A) Quantitative RT-PCR analysis of Il7r mRNA in monocytes (F4/80loCD11bhi) and macrophages (F4/80hiCD11blo) isolated from liver, lung, brain and skin of E14.5 fetuses. Fetal liver macrophage dendritic cell precursors (MDP; CD115+Kit+Flk2–Ly6cCD11b) and adult BM monocytes (CD11b+Gr1mid) were used as positive and negative controls, respectively. Data shown are mean±s.e.m. of ΔΔCT values calculated for Il7r and β-actin and normalized to MDPs (set to 100) across three independent experiments. (B) Quantitative RT-PCR analysis of Il7r mRNA in monocytes (F4/80loCD11bhi) and macrophages (F4/80hiCD11blo) isolated from liver, lung, brain and epidermis (Epi) of neonatal mice at P14. Adult BM Pro-B cells (B220+CD43+) and BM monocytes (CD11b+Gr1+) were used as positive and negative controls, respectively. Data shown are mean±s.e.m. of ΔΔCT values calculated for Il7r and beta-actin and normalized to BM Pro-B cells (set to 100) across three independent experiments. ND, not detected. (C,D) Representative flow cytometric analysis of IL7Rα surface expression in F4/80lo monocytes (CD45+F4/80loCD11bhi; C) and F4/80hi macrophages (CD45+F4/80hiCD11bmid; D) in different tissues at E12.5, E14.5, E17.5, P0 and P14. Gray shaded area indicates FMO control shown for each cell type, respectively. Plots are representative of analysis in five or six mice from three independent experiments. Analysis of common gamma chain expression in fetal tissue macrophages can be found in Fig. S4.

To ascertain the relationship between Il7r-Cre-mediated GFP labeling and IL7Rα surface expression in developing macrophages during fetal development, we profiled surface expression from E12.5 to P14. Considering the high degree of GFP labeling and expression of Il7r mRNA by F4/80lo monocytes during fetal development, we expected to observe monocytes displaying surface IL7R. Unexpectedly, F4/80lo monocytes never displayed detectable IL7Rα surface protein at any of the time points examined (Fig. 4C). Surprisingly, robust IL7Rα surface protein was instead observed on prenatal F4/80hi macrophages in peripheral tissues (lung, brain and skin), whereas minimal surface expression was observed on F4/80hi macrophages in the liver (Fig. 4D). IL7Rα surface protein on peripheral F4/80hi trMacs was observed beginning at E12.5, and peaked at E17.5 (Fig. 4D, middle panel), despite significantly lower GFP labeling compared with F4/80lo monocytes (Fig. 3B,C). The vast majority (>90%) of IL7Rα-expressing macrophages in the brain, lung and skin also co-expressed the common γ chain (CD132; also known as IL2RG; Fig. S5A), indicating expression of a functional IL7 receptor. By P0, macrophages in the liver and lung ceased to express IL7Rα, concomitant with a plateau in GFP labeling by birth (Fig. 3C). In contrast, some proportion of macrophages in the brain and epidermis continued to express IL7Rα protein at the surface postnatally, albeit at lower levels, and IL7Rα surface expression was detectable on epidermal macrophages as late as P14. Continued IL7Rα expression in these particular tissues was consistent with continued postnatal labeling by Il7r-Cre (Fig. 3C) and Il7r mRNA expression (Fig. 4B). These data therefore revealed tightly regulated and highly coordinated expression of IL7Rα in developing macrophages across different tissues.

IL7Rα-expressing monocytes give rise to IL7R+ macrophages ex vivo

We hypothesized that F4/80lo monocytes upregulate Il7r as they exit the liver and migrate to and enter fetal tissues, where they differentiate into F4/80hi macrophages. IL7Rα surface expression is then rapidly switched on as F4/80lo monocytes differentiate into F4/80hi macrophages. To test our hypothesis, we isolated F4/80lo monocytes from the fetal liver at E14.5 and differentiated them into F4/80hi macrophages ex vivo in the presence of macrophage colony-stimulating factor (M-CSF). F4/80loCD11bmid cells were sorted from FL based on Ly6c expression (Fig. 5A) and cultured with 20 ng M-CSF/ml for 5 days. FL monocytes expressed detectable Il7r (Fig. 4A), but IL7R surface expression was not observed (Fig. 5B-C′). M-CSF induced the differentiation of Ly6chiF4/80loCD11bhi to F4/80hi CD11bmid macrophages that expressed CD64 after only 1 day in culture, and macrophage differentiation plateaued at 3 days (Fig. 5C,E,E′). Differentiated F4/80hi macrophages could also be differentiated from F4/80lo monocytes by their larger size and higher cellularity complexity, as defined by forward and side scatter (Fig. S5C′). Remarkably, differentiated F4/80hi cells also upregulated IL7Rα expression on the cell surface (Fig. 5C,C′; also see Fig. S5D). IL7Rα was co-expressed with the common gamma chain (Fig. 5D,D′; Fig. S5D). In contrast to Ly6chi cells, Ly6cloCD11bhi FL monocytes displayed limited differentiation into F4/80hi macrophages, even after 5 days in culture with M-CSF (Fig. S5B). However, additional refinement of these populations by sorting for CD115-expressing cells enhanced macrophage differentiation from Ly6chi F4/80hiCD11bmid monocytes, and also resulted in macrophage differentiation from Ly6cloCD115+F4/80hiCD11bmid cells (Fig. S5C). Both MDP and Ly6clo monocytes displayed significantly higher Cre-mediated labeling compared with Ly6chi monocytes (Fig. S4D), suggesting distinct developmental pathways. These data reveal that Ly6chiCD11bhi cells in the fetal liver that express Il7r (Fig. 4A; Fig. S4E) but not surface IL7R surface protein (Fig. S4F) have the capacity to differentiate into F4/80hi macrophages, and that this differentiation is accompanied by upregulation of surface IL7R expression.

Fig. 5.

Fetalliver monocytes differentiate into IL7R-expressing macrophages ex vivo. (A) Representative gating of F4/80loCD11bhimonocytes (orange gates) from CD11b-enriched FL at E14.5, gated on Ly6c expression (Ly6chi, red; Ly6clo, blue). Adjacent plots represent post-sort purity analysis of FL CD11bhiF4/80lo Ly6chi and Ly6clo monocytes with mean±s.e.m. frequency of sorted populations in triplicate from three independent experiments. (B) Representative FACS plots show surface IL7Rα expression on macrophage (F4/80hi) and monocyte (F4/80loCD11bhi) populations as a function of Ly6c expression, color-coded as gated in A. (C-E′) Representative FACS plots showing gating of monocyte (F4/80loCD11b+) and macrophage (F4/80hiCD11b+) populations and expression of IL7Rα (C), CD132 (D) and CD64 (E) on gated populations following 1, 3 or 5 days of culture of sorted F4/80loCD11bhiLy6chi monocytes with 20 ng m-CSF. Mean fluorescence intensity (MFI; C′,D′,E′) was calculated as the geometric mean minus the FMO for each sample. Gray shaded area indicates FMO control. Values indicate mean frequencies±s.e.m. of gated populations for six to eight different samples in triplicate from three independent experiments. *P<0.05, **P<0.01, ***P<0.001; Student's t-test.

Fig. 5.

Fetalliver monocytes differentiate into IL7R-expressing macrophages ex vivo. (A) Representative gating of F4/80loCD11bhimonocytes (orange gates) from CD11b-enriched FL at E14.5, gated on Ly6c expression (Ly6chi, red; Ly6clo, blue). Adjacent plots represent post-sort purity analysis of FL CD11bhiF4/80lo Ly6chi and Ly6clo monocytes with mean±s.e.m. frequency of sorted populations in triplicate from three independent experiments. (B) Representative FACS plots show surface IL7Rα expression on macrophage (F4/80hi) and monocyte (F4/80loCD11bhi) populations as a function of Ly6c expression, color-coded as gated in A. (C-E′) Representative FACS plots showing gating of monocyte (F4/80loCD11b+) and macrophage (F4/80hiCD11b+) populations and expression of IL7Rα (C), CD132 (D) and CD64 (E) on gated populations following 1, 3 or 5 days of culture of sorted F4/80loCD11bhiLy6chi monocytes with 20 ng m-CSF. Mean fluorescence intensity (MFI; C′,D′,E′) was calculated as the geometric mean minus the FMO for each sample. Gray shaded area indicates FMO control. Values indicate mean frequencies±s.e.m. of gated populations for six to eight different samples in triplicate from three independent experiments. *P<0.05, **P<0.01, ***P<0.001; Student's t-test.

IL7Rα regulates tissue-resident macrophage development

The robust and dynamic expression of IL7R by monocyte precursors and developing tissue macrophages suggested that IL7R regulates fetal trMac development from precursors within fetal tissues. To test this hypothesis, we injected the highly specific IL7Rα monoclonal blocking antibody A7R34, or an IgG2A control, into pregnant mice during fetal development (Fig. 6A). Injection of A7R34 during pregnancy completely blocks IL7R signaling, as evidenced by deletion of Peyer's patches in developing embryos following a single injection (Hashizume et al., 2008; Yoshida et al., 1999). We injected pregnant WT mice with A7R34 or IgG2A control at E13.5 and E15.5, coincident with robust IL7Rα expression on fetal macrophages (Fig. 4B), and examined cellularity of macrophages in the epidermis, lung, liver and brain in neonates 9 days later (Fig. 6A). Macrophage cellularity in the epidermis, liver and lung was significantly reduced following this temporally limited blockade of IL7Rα signaling, whereas microglia were unchanged (Fig. 6B).

Fig. 6.

IL7R regulates tissue-resident macrophage development. (A) Schematic illustrating the timeline for injection and analysis of the IL7R blocking antibody, A7R43, and IgG2A control. Timed-mated WT mice were injected at E13.5 and E15.5 with 600 mg of the IL7Rα blocking antibody or equivalent control injection. Pups were analyzed 9 days after final injection and cellularity of macrophages in the liver, lung, brain and epidermis (Epi) were determined. (B) Representative FACS plots and associated quantification of cells/mg of tissue macrophages (live CD45+F4/80hiCD11blo) in the liver, lung, brain and epidermis of neonates examined 9 days following maternal injections of the A7R43 IL7Rα blocking antibody or IgG2A control administered at E13.5 and E15.5. n=8-11 representing three independent experiments. *P<0.05. (C) Representative FACS plots and associated quantification of cells/mg of tissue macrophages (live CD45+F4/80hiCD11blo) in the liver, lung, brain and epidermis of Il7r−/− and WT neonates at P19. n=4 representing four independent experiments. *P<0.05. (D) A model for dynamic IL7Rα expression during fetal macrophage development. Fetal liver F4/80lo monocytes express Il7r message, and mRNA expression is upregulated as F4/80lo monocytes exit the liver and transit to peripheral tissues. However, upregulation of message is not associated with measurable surface expression in F4/80lo monocytes. IL7Rα surface expression only occurs upon differentiation of monocytes into F4/80hi macrophages in the tissues, at which point message levels dissipate. Continued message and surface expression postnatally within microglia and LCs account for higher levels of GFP labeling in those tissues, whereas GFP labeling is equilibrated by birth in the AMs of the lung and KCs of the liver.

Fig. 6.

IL7R regulates tissue-resident macrophage development. (A) Schematic illustrating the timeline for injection and analysis of the IL7R blocking antibody, A7R43, and IgG2A control. Timed-mated WT mice were injected at E13.5 and E15.5 with 600 mg of the IL7Rα blocking antibody or equivalent control injection. Pups were analyzed 9 days after final injection and cellularity of macrophages in the liver, lung, brain and epidermis (Epi) were determined. (B) Representative FACS plots and associated quantification of cells/mg of tissue macrophages (live CD45+F4/80hiCD11blo) in the liver, lung, brain and epidermis of neonates examined 9 days following maternal injections of the A7R43 IL7Rα blocking antibody or IgG2A control administered at E13.5 and E15.5. n=8-11 representing three independent experiments. *P<0.05. (C) Representative FACS plots and associated quantification of cells/mg of tissue macrophages (live CD45+F4/80hiCD11blo) in the liver, lung, brain and epidermis of Il7r−/− and WT neonates at P19. n=4 representing four independent experiments. *P<0.05. (D) A model for dynamic IL7Rα expression during fetal macrophage development. Fetal liver F4/80lo monocytes express Il7r message, and mRNA expression is upregulated as F4/80lo monocytes exit the liver and transit to peripheral tissues. However, upregulation of message is not associated with measurable surface expression in F4/80lo monocytes. IL7Rα surface expression only occurs upon differentiation of monocytes into F4/80hi macrophages in the tissues, at which point message levels dissipate. Continued message and surface expression postnatally within microglia and LCs account for higher levels of GFP labeling in those tissues, whereas GFP labeling is equilibrated by birth in the AMs of the lung and KCs of the liver.

We further investigated trMac cellularity in Il7r−/− mice. Although Il7r deletion did not affect YS hematopoiesis (Fig. 2), examination at P19 revealed that Il7r deletion drastically reduced cellularity of trMacs in all tissues (Fig. 6C, Fig. S6A). Il7r deletion did not affect cellularity of other tissue myeloid populations, such as neutrophils (Fig. S6B). By adulthood, differences in macrophage cellularity had normalized (Fig. S6C), despite sustained impairments in lymphocyte development (Fig. S6C′). Cellularity of trMacs was similarly normalized by adulthood following developmental IL7R blockade (Fig. S6D), as were B cells (Fig. S6D′). As trMacs are capable of self-maintenance across adulthood, these data suggest that IL7R plays a unique role in the establishment of these populations, but is not required for their homeostatic maintenance in adulthood. Together with the IL7R blocking experiments, these data indicate that IL7R plays a unique role in the establishment of trMacs during perinatal development.

DISCUSSION

Our investigation has revealed a completely novel role for IL7R in fetal myeloid development, and specifically in the generation of fetal-specified trMacs. Tracing the progeny of cells mapped by the lymphoid marker IL7Rα revealed that trMacs derived from fetal hematopoiesis were distinctly marked by IL7Rα expression history (Fig. 1). Our comprehensive analysis of Il7r-Cre labeling, Il7r mRNA levels, and surface expression concluded that trMac labeling was not due solely to derivation from an IL7Rα-labeled bipotent progenitor, nor was labeling acquired in adulthood. Instead, our developmental analysis revealed that labeling of trMacs by Il7r-Cre occurred as a result of dynamic stage- and tissue-specific expression of IL7R during macrophage development within tissues during fetal development (Figs 3 and 4). We showed that the CD11bhiLy6chi fetal liver monocytes rapidly upregulated surface IL7R as they differentiated into F4/80hi macrophages ex vivo (Fig. 5), and observed robust IL7R surface expression on developing macrophages during a limited window of fetal development. Using two different loss-of-function approaches, we further demonstrated that blocking IL7R signaling during the window of robust IL7R expression by developing trMacs impairs their establishment (Fig. 6). In adult BM hematopoiesis, IL7Rα expression exclusively marks lymphoid cells (Schlenner et al., 2010), and the many functions of IL7R signaling in lymphopoiesis are well-delineated (Fry and Mackall, 2005), including proliferation, differentiation and survival. IL7R may therefore regulate similar processes in developing macrophages, but the mechanisms and signaling pathways for IL7R in myeloid cells remain to be established. By revealing a role for IL7R in trMac development, our data identify a function of IL7R during fetal development that extends beyond regulation of the lymphoid lineage, contributing to accumulating evidence that fetal hematopoietic lineage specification is considerably less constrained than adult hematopoiesis (Beaudin et al., 2016; Mebius et al., 2001; Notta et al., 2016).

Common signaling factors, including dependency on the transcription factor PU.1 (also known as SPI1) (Schulz et al., 2012) and signaling downstream of the CSF-1 receptor (Chitu and Stanley, 2017), regulate macrophage development across multiple tissues, whereas other signals regulate and maintain tissue-specific identity and function (Guilliams et al., 2013; Mass et al., 2016; Scott et al., 2018). IL7R appears to function as a regulatory factor across different tissues, but may also have distinct roles in different tissues across ontogeny. Blocking IL7Rα function during a narrow fetal window (E13.5-E15.5) negatively affected cellularity of liver, lung and epidermal macrophages, with no effect on microglia (Fig. 6B). In contrast, germline deletion of IL7Rα in the Il7r−/− mouse significantly impaired trMac cellularity across all tissues, including microglia, examined at P19 (Fig. 6C). The different effect of total knockout and transient blockade of IL7Rα on trMac development suggests that the window of dependency on IL7R signaling differs between different tissues. The requirement for IL7Rα during postnatal microglia development fits with recent reports of rapid changes in proliferation and apoptosis occurring postnatally in microglia (Askew et al., 2017; Nikodemova et al., 2015), and suggests the possibility that IL7Rα contributes to proliferation or survival of microglia in the transition between the postnatal period and adulthood. Although IL7Rα is not expressed by adult myeloid cells at homeostasis (Schlenner et al., 2010), there have been a handful of reports suggesting that IL7 can promote monocyte activation in the context of autoimmunity and infection (Chen et al., 2013; Gessner et al., 1993), and a bipotent IL7R+ lympho-myeloid progenitor has been identified that emerges in the context of malaria infection (Belyaev et al., 2010). Whether autoimmunity or other inflammatory conditions can promote the usage of fetal hematopoietic differentiation pathways to confer distinct functions on myeloid cells during disease states is an interesting hypothesis that remains be tested experimentally.

We used three different lineage-tracing models based on lymphoid markers associated with both increased lymphoid potential (Flk2, Rag1, IL7Rα; Fig. 1) (Boyer et al., 2019; Forsberg et al., 2006; Igarashi et al., 2002; Kondo et al., 1997) and overlapping expression in FL progenitors (Beaudin et al., 2016; Boiers et al., 2013) to trace trMac origin. The stark contrast in labeling between these models allows us to exclude the possibility that labeling of adult trMacs by Il7r-Cre reflected inheritance from an earlier lymphomyeloid progenitor. A clear example is epidermal LCs: if labeling of LCs was due to derivation from lymphomyeloid progenitors, LCs would be robustly labeled not only by Il7r-Cre, but also by Flk2-Cre and Rag1-Cre, as those genes are expressed in IL7R+ FL progenitors (Boiers et al., 2013). The high degree of Il7r-Cre labeling of trMacs was similarly not reflected in YS EMPs or YS macrophages (Fig. 2, Fig. S2) and IL7R deletion had no effect on YS EMP cellularity or the development of YS macrophages during embryonic hematopoiesis (Fig. 2D,E), but resulted in significant impairments later in trMac development (Fig. 6C). Together, these data suggest that although IL7R expression labels a multipotent fetal liver progenitor, expression of IL7R on developing tissues macrophages later in gestation regulates their development during later waves of macrophage seeding (De et al., 2018; Ferrero et al., 2018; Tan and Krasnow, 2016).

Our analysis of the IL7R knockout mouse and in response to physiological blockade of IL7R reveal IL7R as a novel regulator of tissue macrophage development. Functional inhibition of IL7R also affected lymphocyte cellularity (Fig. S6C′), as previously reported (Hashizume et al., 2008; Peschon et al., 1994; Yoshida et al., 1999). One caveat is that the impairments in trMac development may be secondary to impaired lymphocyte development. However, despite sustained impairments in lymphocyte development in the IL7R knockout (Fig. S6C′), trMac cellularity recovered by adulthood (Fig. S6C), arguing that lymphocyte impairment alone is not responsible for impaired trMac development. Deletion of IL7R had no effect on YS EMPs or YS macrophage generation, affirming that IL7R plays a more crucial role in fetal macrophage generation at later stages of hematopoiesis.

Fetal F4/80hi macrophages expressed negligible Il7r message levels (Fig. 4A) yet expressed robust surface IL7R protein (Fig. 4D) and acquired Il7r-Cre labeling across fetal development (Fig. 3C). In lung and liver, for example, increased GFP labeling in F4/80hi trMacs across fetal development did not reflect Cre-driven reporter expression, as trMacs did not express Il7r during fetal development (Fig. 4A). The most parsimonious explanation for this surprising discrepancy is the initiation of Cre recombination in IL7R-marked F4/80lo macrophage precursors with subsequent inheritance of the floxed allele by F4/80hi trMacs upon differentiation. F4/80hi trMac differentiation is then coincident with the rapid translation of Il7r message into IL7Rα protein and surface display (Fig. 6D). Indeed, our ex vivo differentiation assay revealed that Ly6c+ FL monocytes expressing Il7r mRNA (Fig. 4C; Fig. S4E) differentiate into F4/80hi macrophages that upregulate surface IL7R protein ex vivo (Fig. 5). In vivo, the rapid and dynamic regulation of IL7R protein surface expression may reflect a response to ligand in tissues, as surface expression of IL7R is regulated by IL7 availability (Clark et al., 2014; Wei et al., 2000). The lower surface expression of IL7R on fetal liver F4/80hi macrophages compared with peripheral tissues (Fig. 4D) in vivo may also reflect surface regulation by immediate sources of IL7 ligand in the fetal liver (Namen et al., 1988).

By tracking GFP expression within fetal myeloid cells in the Il7r-Cre model, we observed the contribution or replacement of GFP-labeled F4/80lo precursors to F4/80hi trMacs in the lung and liver, as evidenced by increased labeling in F4/80hi macrophages across ontogeny (Fig. 3C). Whereas trMacs of the lung and liver exhibited robust labeling (∼40%) that had plateaued at birth (Fig. 3A-C), Il7r mRNA expression (Fig. 4B,D; Mass et al., 2016) and Cre-mediated labeling of LC and microglia continued postnatally, leading to almost complete labeling by Il7r-Cre in adulthood. Although monocytes entering the epidermis and brain expressed both the highest levels of Il7r mRNA and had the greatest degree of Cre-mediated labeling, there was very little monocyte infiltration from E14.5 and beyond (Fig. 4A). Instead, the higher degree of labeling in the epidermis and brain could be attributed to higher and persistent expression of IL7Rα within trMacs peri- and postnatally, leading to increased GFP labeling (Fig. 3). Together, our analysis provides additional support for the contribution of FL F4/80lo macrophage precursors to specific trMac compartments during late gestation (Guilliams et al., 2013; Hoeffel et al., 2015; Rantakari et al., 2016; Tan and Krasnow, 2016), and reveals how IL7R expression during macrophage differentiation regulates establishment of tissue macrophage compartments.

MATERIALS AND METHODS

Mice

All animals were housed and bred in the AALAC accredited vivaria at UC Santa Cruz or UC Merced and group housed in ventilated cages on a standard 12:12 light cycle. All procedures were approved by the UCSC or the UC Merced Institutional Animal Care and Use (IACUC) committees. IL7Rα-Cre (Schlenner et al., 2010), Rag1-Cre (Welner et al., 2009) and Flk2-Cre (Benz et al., 2008) mice, obtained under fully executed Material Transfer Agreements, were crossed to homozygous Rosa26mTmG females (Muzumdar et al., 2007) to generate ‘switch’ lines, all on the C57Bl/6 background. WT C56Bl/6 mice were used for controls and for all expression experiments. Adult male and female mice were used randomly and indiscriminately, with the exception of the FlkSwitch line, for which only males were used because of their high and uniform floxing efficiency. Similarly, mice for developmental analysis were used indiscriminately without knowledge of gender.

Tissue and cell isolation

Mice were sacrificed by CO2 inhalation. Gravid uteri removed, and individual embryos dissected. Adult liver, lung, brain and skin were isolated and treated with 1× PBS (+/+) with 2% serum, 0.2-1 mg/ml collagenase IV (Gibco) with or without 100 U/ml DNase1 for 20 min to 2 h. For adult epidermis isolation, ears were first incubated with 1× PBS (+/+) containing 2.4 mg/ml dispase (Gibco) to separate the epidermis, followed by 2 h incubation of the epidermis with 1/mg/ml collagenase. Following incubation, all tissues were passaged through a 16 g needle or a 19 g needle ten times, and then filtered through a 70 µm filter.

Flow cytometry

Cell labeling was performed on ice in 1× PBS with 5 mM EDTA and 2% serum. Antibodies used are listed in Table S1. Analysis was performed on BD FACS Aria II at University of California-Santa Cruz, and BD FACS Aria III and the University of California-Merced and analyzed using FlowJo.

Transplantation assays

Transplantation assays were performed as previously described (Beaudin et al., 2014; Beaudin et al., 2016; Smith-Berdan et al., 2015; Ugarte et al., 2015; Boyer et al., 2019). Briefly, sorted Tom+ or GFP+ KLS cells were isolated from IL7RαSwitch or Rag1Switch E14.5 fetal liver donors. WT recipient mice aged 8-12 weeks were sublethally irradiated (750 rad, single dose). Under isofluorane-induced general anesthesia, sorted cells were transplanted intravenously. Recipient mice were bled at 4, 8, 12 and 16 weeks post-transplantation via the tail vein and peripheral blood was analyzed for donor chimerism by means of fluorescence profiles and antibodies to lineage markers. Long-term multilineage reconstitution was defined as chimerism in both the lymphoid and myeloid lineages of >0.1% at 16 weeks post-transplantation.

Ex vivo macrophage culture

E14.5 fetal livers from WT embryos were harvested and homogenized via trituration. CD11b+ cells were enriched by positive selection with CD11b biotin-conjugated antibodies and streptavidin microbeads, using LS columns (Miltenyi). F4/80lo CD11bmid cells were sorted based on Ly6c expression or CD115 expression and cultured in a 96-well plate (DMEM, 20% FBS, 1 mM sodium pyruvate, 10 mM HEPES, 0.1 mM 2-mercaptoethanol and 50 mg/ml primocin, Life Technologies). Cells were cultured in triplicate in the presence of 20 ng/ml M-CSF and analyzed at three different time points (days 1, 3 and 5) for the presence of F4/80hi CD11bmid macrophages and for surface expression of IL7R, common gamma chain (CD132) and CD64.

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR) analysis

RNA isolation from sorted cells was accomplished using the RNeasy mini kit (Qiagen), and cDNA was reverse transcribed from RNA (High capacity cDNA reverse transcription kit, ThermoFisher Scientific). TaqMan probes (TaqMan Gene Expression Assay, ThermoFisher Scientific) were used for qRT-PCR analysis on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific) in comparative CT mode. Samples were run in triplicate and were run with positive (CD43+ B220+ Pro B-cells), negative (Gr1+ CD11b+ BM monocytes), and no cDNA controls.

Quantification and statistical analysis

Number of experiments, n, and what n represents can be found in the legend for each figure. Statistical significance was determined by two-tailed unpaired Student's t-test. All data are shown as mean±s.e.m. representing at least three independent experiments.

Acknowledgements

We thank Drs Hans-Reimer Rodewald and Susan M. Schlenner for the IL7RαCre strain; Drs Patricia Ernst and Terence Rabbitts for the Rag1Cre strain; Dr T. Boehm for the Flt3Cre strain; Bari Nazario and the UCSC Institute for the Biology of Stem Cells for flow cytometry support; and David Gravano and the UC Merced Stem Cell Instrumentation Foundry for flow cytometry support. CIRM Facilities awards CL1-00506 and FA1-00617-1 to UCSC.

Footnotes

Author contributions

Conceptualization: A.E.B., E.C.F.; Methodology: A.E.B., E.C.F.; Formal analysis: E.C.F.; Investigation: G.A.L., T.C., C.H.V., A.W., A.E.B.; Resources: E.C.F.; Writing - original draft: A.E.B., E.C.F.; Writing - review & editing: G.A.L., T.C., A.W.; Visualization: A.E.B., E.C.F.; Supervision: A.E.B., E.C.F.; Funding acquisition: E.C.F.

Funding

This work was supported by a National Institute of Diabetes and Digestive and Kidney Diseases award (R01DK100917), an Alex's Lemonade Stand Foundation for Childhood Cancer Innovation Award (SC-20130752), and an American Asthma Foundation Research Scholar award (A16-0525) to E.C.F.; by a National Heart, Lung, and Blood Institute (NHLBI) award (R01HL140781) to A.E.B.; by California Institute for Regenerative Medicine (CIRM) SCILL grant (TB1-01195) to T.C. via San Jose State University. E.C.F. is the recipient of a CIRM New Faculty Award (RN1-00540) and an American Cancer Society Research Scholar Award (RSG-13-193-01-DDC); A.E.B. is the recipient of an NHLBI Mentored Career Development Award (K01HL130753). Deposited in PMC for release after 12 months.

References

Adolfsson
,
J.
,
Månsson
,
R.
,
Buza-Vidas
,
N.
,
Hultquist
,
A.
,
Liuba
,
K.
,
Jensen
,
C. T.
,
Bryder
,
D.
,
Yang
,
L.
,
Borge
,
O.-J.
,
Thoren
,
L. A. M.
, et al. 
(
2005
).
Identification of Flt3+ lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment
.
Cell
121
,
295
-
306
.
Ajami
,
B.
,
Bennett
,
J. L.
,
Krieger
,
C.
,
Tetzlaff
,
W.
and
Rossi
,
F. M. V.
(
2007
).
Local self-renewal can sustain CNS microglia maintenance and function throughout adult life
.
Nat. Neurosci.
10
,
1538
-
1543
.
Askew
,
K.
,
Li
,
K.
,
Olmos-Alonso
,
A.
,
Garcia-Moreno
,
F.
,
Liang
,
Y.
,
Richardson
,
P.
,
Tipton
,
T.
,
Chapman
,
M. A.
,
Riecken
,
K.
,
Beccari
,
S.
, et al. 
(
2017
).
Coupled proliferation and apoptosis maintain the rapid turnover of microglia in the adult brain
.
Cell Rep.
18
,
391
-
405
.
Beaudin
,
A. E.
and
Forsberg
,
E. C.
(
2016
).
To B1a or not to B1a: do hematopoietic stem cells contribute to tissue-resident immune cells?
Blood
128
,
2765
-
2769
.
Beaudin
,
A. E.
,
Boyer
,
S. W.
and
Forsberg
,
E. C.
(
2014
).
Flk2/Flt3 promotes both myeloid and lymphoid development by expanding non-self-renewing multipotent hematopoietic progenitor cells
.
Exp. Hematol.
42
,
218
-
229.e214
.
Beaudin
,
A. E.
,
Boyer
,
S. W.
,
Perez-Cunningham
,
J.
,
Hernandez
,
G. E.
,
Derderian
,
S. C.
,
Jujjavarapu
,
C.
,
Aaserude
,
E.
,
MacKenzie
,
T.
and
Forsberg
,
E. C.
(
2016
).
A transient developmental hematopoietic stem cell gives rise to innate-like B and T cells
.
Cell Stem Cell
19
,
768
-
783
.
Belyaev
,
N. N.
,
Brown
,
D. E.
,
Diaz
,
A.-I. G.
,
Rae
,
A.
,
Jarra
,
W.
,
Thompson
,
J.
,
Langhorne
,
J.
and
Potocnik
,
A. J.
(
2010
).
Induction of an IL7-R(+)c-Kit(hi) myelolymphoid progenitor critically dependent on IFN-gamma signaling during acute malaria
.
Nat. Immunol.
11
,
477
-
485
.
Benz
,
C.
,
Martins
,
V. C.
,
Radtke
,
F.
and
Bleul
,
C. C.
(
2008
).
The stream of precursors that colonizes the thymus proceeds selectively through the early T lineage precursor stage of T cell development
.
J. Exp. Med.
205
,
1187
-
1199
.
Boiers
,
C.
,
Carrelha
,
J.
,
Lutteropp
,
M.
,
Luc
,
S.
,
Green
,
J. C. A.
,
Azzoni
,
E.
,
Woll
,
P. S.
,
Mead
,
A. J.
,
Hultquist
,
A.
,
Swiers
,
G.
, et al. 
(
2013
).
Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells
.
Cell Stem Cell
13
,
535
-
548
.
Boyer
,
S. W.
,
Schroeder
,
A. V.
,
Smith-Berdan
,
S.
and
Forsberg
,
E. C.
(
2011
).
All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells
.
Cell Stem Cell
9
,
64
-
73
.
Boyer
,
S. W.
,
Beaudin
,
A. E.
and
Forsberg
,
E. C.
(
2012
).
Mapping differentiation pathways from hematopoietic stem cells using Flk2/Flt3 lineage tracing
.
Cell Cycle
11
,
3180
-
3188
.
Boyer
,
S. W.
,
Rajendiran
,
S.
,
Beaudin
,
A. E.
,
Smith-Berdan
,
S.
,
Muthuswamy
,
P. K.
,
Perez-Cunningham
,
J.
,
Martin
,
E. W.
,
Cheung
,
C.
,
Tsang
,
H.
,
Landon
,
M.
, et al. 
(
2019
).
Clonal and quantitative in vivo assessment of hematopoietic stem cell differentiation reveals strong erythroid potential of multipotent cells
.
Stem Cell Rep.
12
,
801
-
815
.
Chen
,
Z.
,
Kim
,
S.-J.
,
Chamberlain
,
N. D.
,
Pickens
,
S. R.
,
Volin
,
M. V.
,
Volkov
,
S.
,
Arami
,
S.
,
Christman
,
J. W.
,
Prabhakar
,
B. S.
,
Swedler
,
W.
, et al. 
(
2013
).
The novel role of IL-7 ligation to IL-7 receptor in myeloid cells of rheumatoid arthritis and collagen-induced arthritis
.
J. Immunol.
190
,
5256
-
5266
.
Chitu
,
V.
and
Stanley
,
E. R.
(
2017
).
Regulation of embryonic and postnatal development by the CSF-1 receptor
.
Curr. Top. Dev. Biol.
123
,
229
-
275
.
Clark
,
M. R.
,
Mandal
,
M.
,
Ochiai
,
K.
and
Singh
,
H.
(
2014
).
Orchestrating B cell lymphopoiesis through interplay of IL-7 receptor and pre-B cell receptor signalling
.
Nat. Rev. Immunol.
14
,
69
-
80
.
Cool
,
T.
and
Forsberg
,
E. C.
(
2019
).
Chasing Mavericks: the quest for defining developmental waves of hematopoiesis
.
Curr. Top. Dev. Biol.
132
,
1
-
29
.
De
,
S.
,
Van Deren
,
D.
,
Peden
,
E.
,
Hockin
,
M.
,
Boulet
,
A.
,
Titen
,
S.
and
Capecchi
,
M. R.
(
2018
).
Two distinct ontogenies confer heterogeneity to mouse brain microglia
.
Development
145
,
dev152306
.
Epelman
,
S.
,
Lavine
,
K. J.
,
Beaudin
,
A. E.
,
Sojka
,
D. K.
,
Carrero
,
J. A.
,
Calderon
,
B.
,
Brija
,
T.
,
Gautier
,
E. L.
,
Ivanov
,
S.
,
Satpathy
,
A. T.
, et al. 
(
2014
).
Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation
.
Immunity
40
,
91
-
104
.
Ferrero
,
G.
,
Mahony
,
C. B.
,
Dupuis
,
E.
,
Yvernogeau
,
L.
,
Di Ruggiero
,
E.
,
Miserocchi
,
M.
,
Caron
,
M.
,
Robin
,
C.
,
Traver
,
D.
,
Bertrand
,
J. Y.
, et al. 
(
2018
).
Embryonic microglia derive from primitive macrophages and are replaced by cmyb-dependent definitive microglia in zebrafish
.
Cell Rep.
24
,
130
-
141
.
Forsberg
,
E. C.
,
Serwold
,
T.
,
Kogan
,
S.
,
Weissman
,
I. L.
and
Passegué
,
E.
(
2006
).
New evidence supporting megakaryocyte-erythrocyte potential of flk2/flt3+ multipotent hematopoietic progenitors
.
Cell
126
,
415
-
426
.
Fry
,
T. J.
and
Mackall
,
C. L.
(
2005
).
The many faces of IL-7: from lymphopoiesis to peripheral T cell maintenance
.
J. Immunol.
174
,
6571
-
6576
.
Gessner
,
A.
,
Vieth
,
M.
,
Will
,
A.
,
Schröppel
,
K.
and
Röllinghoff
,
M.
(
1993
).
Interleukin-7 enhances antimicrobial activity against Leishmania major in murine macrophages
.
Infect. Immun.
61
,
4008
-
4012
.
Ginhoux
,
F.
,
Greter
,
M.
,
Leboeuf
,
M.
,
Nandi
,
S.
,
See
,
P.
,
Gokhan
,
S.
,
Mehler
,
M. F.
,
Conway
,
S. J.
,
Ng
,
L. G.
,
Stanley
,
E. R.
, et al. 
(
2010
).
Fate mapping analysis reveals that adult microglia derive from primitive macrophages
.
Science
330
,
841
-
845
.
Gomez Perdiguero
,
E.
,
Klapproth
,
K.
,
Schulz
,
C.
,
Busch
,
K.
,
Azzoni
,
E.
,
Crozet
,
L.
,
Garner
,
H.
,
Trouillet
,
C.
,
de Bruijn
,
M. F.
,
Geissmann
,
F.
, et al. 
(
2015
).
Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors
.
Nature
518
,
547
-
551
.
Guilliams
,
M.
,
De Kleer
,
I.
,
Henri
,
S.
,
Post
,
S.
,
Vanhoutte
,
L.
,
De Prijck
,
S.
,
Deswarte
,
K.
,
Malissen
,
B.
,
Hammad
,
H.
and
Lambrecht
,
B. N.
(
2013
).
Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF
.
J. Exp. Med.
210
,
1977
-
1992
.
Hashimoto
,
D.
,
Chow
,
A.
,
Noizat
,
C.
,
Teo
,
P.
,
Beasley
,
M. B.
,
Leboeuf
,
M.
,
Becker
,
C. D.
,
See
,
P.
,
Price
,
J.
,
Lucas
,
D.
, et al. 
(
2013
).
Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes
.
Immunity
38
,
792
-
804
.
Hashizume
,
T.
,
Togawa
,
A.
,
Nochi
,
T.
,
Igarashi
,
O.
,
Kweon
,
M.-N.
,
Kiyono
,
H.
and
Yamamoto
,
M.
(
2008
).
Peyer's patches are required for intestinal immunoglobulin A responses to Salmonella spp
.
Infect. Immun.
76
,
927
-
934
.
Hoeffel
,
G.
,
Wang
,
Y.
,
Greter
,
M.
,
See
,
P.
,
Teo
,
P.
,
Malleret
,
B.
,
Leboeuf
,
M.
,
Low
,
D.
,
Oller
,
G.
,
Almeida
,
F.
, et al. 
(
2012
).
Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages
.
J. Exp. Med.
209
,
1167
-
1181
.
Hoeffel
,
G.
,
Chen
,
J.
,
Lavin
,
Y.
,
Low
,
D.
,
Almeida
,
F. F.
,
See
,
P.
,
Beaudin
,
A. E.
,
Lum
,
J.
,
Low
,
I.
,
Forsberg
,
E. C.
, et al. 
(
2015
).
C-myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages
.
Immunity
42
,
665
-
678
.
Hulsmans
,
M.
,
Clauss
,
S.
,
Xiao
,
L.
,
Aguirre
,
A. D.
,
King
,
K. R.
,
Hanley
,
A.
,
Hucker
,
W. J.
,
Wülfers
,
E. M.
,
Seemann
,
G.
,
Courties
,
G.
, et al. 
(
2017
).
Macrophages facilitate electrical conduction in the heart
.
Cell
169
,
510
-
522.e20
.
Igarashi
,
H.
,
Gregory
,
S. C.
,
Yokota
,
T.
,
Sakaguchi
,
N.
and
Kincade
,
P. W.
(
2002
).
Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow
.
Immunity
17
,
117
-
130
.
Kierdorf
,
K.
,
Erny
,
D.
,
Goldmann
,
T.
,
Sander
,
V.
,
Schulz
,
C.
,
Perdiguero
,
E. G.
,
Wieghofer
,
P.
,
Heinrich
,
A.
,
Riemke
,
P.
,
Hölscher
,
C.
, et al. 
(
2013
).
Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways
.
Nat. Neurosci.
16
,
273
-
280
.
Kondo
,
M.
,
Weissman
,
I. L.
and
Akashi
,
K.
(
1997
).
Identification of clonogenic common lymphoid progenitors in mouse bone marrow
.
Cell
91
,
661
-
672
.
Mass
,
E.
,
Ballesteros
,
I.
,
Farlik
,
M.
,
Halbritter
,
F.
,
Günther
,
P.
,
Crozet
,
L.
,
Jacome-Galarza
,
C. E.
,
Händler
,
K.
,
Klughammer
,
J.
,
Kobayashi
,
Y.
, et al. 
(
2016
).
Specification of tissue-resident macrophages during organogenesis
.
Science
353
,
aaf4238
.
Mebius
,
R. E.
,
Miyamoto
,
T.
,
Christensen
,
J.
,
Domen
,
J.
,
Cupedo
,
T.
,
Weissman
,
I. L.
and
Akashi
,
K.
(
2001
).
The fetal liver counterpart of adult common lymphoid progenitors gives rise to all lymphoid lineages, CD45+CD4+CD3− cells, as well as macrophages
.
J. Immunol.
166
,
6593
-
6601
.
Merad
,
M.
,
Manz
,
M. G.
,
Karsunky
,
H.
,
Wagers
,
A.
,
Peters
,
W.
,
Charo
,
I.
,
Weissman
,
I. L.
,
Cyster
,
J. G.
and
Engleman
,
E. G.
(
2002
).
Langerhans cells renew in the skin throughout life under steady-state conditions
.
Nat. Immunol.
3
,
1135
-
1141
.
Muzumdar
,
M. D.
,
Tasic
,
B.
,
Miyamichi
,
K.
,
Li
,
L.
and
Luo
,
L.
(
2007
).
A global double-fluorescent Cre reporter mouse
.
Genesis
45
,
593
-
605
.
Namen
,
A. E.
,
Lupton
,
S.
,
Hjerrild
,
K.
,
Wignall
,
J.
,
Mochizuki
,
D. Y.
,
Schmierer
,
A.
,
Mosley
,
B.
,
March
,
C. J.
,
Urdal
,
D.
and
Gillis
,
S.
(
1988
).
Stimulation of B-cell progenitors by cloned murine interleukin-7
.
Nature
333
,
571
-
573
.
Nikodemova
,
M.
,
Kimyon
,
R. S.
,
De
,
I.
,
Small
,
A. L.
,
Collier
,
L. S.
and
Watters
,
J. J.
(
2015
).
Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week
.
J. Neuroimmunol.
278
,
280
-
288
.
Notta
,
F.
,
Zandi
,
S.
,
Takayama
,
N.
,
Dobson
,
S.
,
Gan
,
O. I.
,
Wilson
,
G.
,
Kaufmann
,
K. B.
,
McLeod
,
J.
,
Laurenti
,
E.
,
Dunant
,
C. F.
, et al. 
(
2016
).
Distinct routes of lineage development reshape the human blood hierarchy across ontogeny
.
Science
351
,
aab2116
.
Peschon
,
J. J.
,
Morrissey
,
P. J.
,
Grabstein
,
K. H.
,
Ramsdell
,
F. J.
,
Maraskovsky
,
E.
,
Gliniak
,
B. C.
,
Park
,
L. S.
,
Ziegler
,
S. F.
,
Williams
,
D. E.
,
Ware
,
C. B.
, et al. 
(
1994
).
Early lymphocyte expansion is severely impaired in interleukin 7 receptor-deficient mice
.
J. Exp. Med.
180
,
1955
-
1960
.
Rantakari
,
P.
,
Jäppinen
,
N.
,
Lokka
,
E.
,
Mokkala
,
E.
,
Gerke
,
H.
,
Peuhu
,
E.
,
Ivaska
,
J.
,
Elima
,
K.
,
Auvinen
,
K.
and
Salmi
,
M.
(
2016
).
Fetal liver endothelium regulates the seeding of tissue-resident macrophages
.
Nature
538
,
392
-
396
.
Sawai
,
C. M.
,
Babovic
,
S.
,
Upadhaya
,
S.
,
Knapp
,
D. J. H. F.
,
Lavin
,
Y.
,
Lau
,
C. M.
,
Goloborodko
,
A.
,
Feng
,
J.
,
Fujisaki
,
J.
,
Ding
,
L.
, et al. 
(
2016
).
Hematopoietic stem cells are the major source of multilineage hematopoiesis in adult animals
.
Immunity
45
,
597
-
609
.
Schlenner
,
S. M.
,
Madan
,
V.
,
Busch
,
K.
,
Tietz
,
A.
,
Läufle
,
C.
,
Costa
,
C.
,
Blum
,
C.
,
Fehling
,
H. J.
and
Rodewald
,
H.-R.
(
2010
).
Fate mapping reveals separate origins of T cells and myeloid lineages in the thymus
.
Immunity
32
,
426
-
436
.
Schulz
,
C.
,
Gomez Perdiguero
,
E.
,
Chorro
,
L.
,
Szabo-Rogers
,
H.
,
Cagnard
,
N.
,
Kierdorf
,
K.
,
Prinz
,
M.
,
Wu
,
B.
,
Jacobsen
,
S. E. W.
,
Pollard
,
J. W.
, et al. 
(
2012
).
A lineage of myeloid cells independent of Myb and hematopoietic stem cells
.
Science
336
,
86
-
90
.
Scott
,
C. L.
,
T'Jonck
,
W.
,
Martens
,
L.
,
Todorov
,
H.
,
Sichien
,
D.
,
Soen
,
B.
,
Bonnardel
,
J.
,
De Prijck
,
S.
,
Vandamme
,
N.
,
Cannoodt
,
R.
, et al. 
(
2018
).
The transcription factor ZEB2 is required to maintain the tissue-specific identities of macrophages
.
Immunity
49
,
312
-
325.e315
.
Sheng
,
J.
,
Ruedl
,
C.
and
Karjalainen
,
K.
(
2015
).
Most tissue-resident macrophages except microglia are derived from fetal hematopoietic stem cells
.
Immunity
43
,
382
-
393
.
Smith-Berdan
,
S.
,
Nguyen
,
A.
,
Hong
,
M. A.
and
Forsberg
,
E. C.
(
2015
).
ROBO4-mediated vascular integrity regulates the directionality of hematopoietic stem cell trafficking
.
Stem Cell Rep.
4
,
255
-
268
.
Stremmel
,
C.
,
Schuchert
,
R.
,
Wagner
,
F.
,
Thaler
,
R.
,
Weinberger
,
T.
,
Pick
,
R.
,
Mass
,
E.
,
Ishikawa-Ankerhold
,
H. C.
,
Margraf
,
A.
,
Hutter
,
S.
, et al. 
(
2018
).
Yolk sac macrophage progenitors traffic to the embryo during defined stages of development
.
Nat. Commun.
9
,
75
.
Tan
,
S. Y. S.
and
Krasnow
,
M. A.
(
2016
).
Developmental origin of lung macrophage diversity
.
Development
143
,
1318
-
1327
.
Ugarte
,
F.
,
Sousae
,
R.
,
Cinquin
,
B.
,
Martin
,
E. W.
,
Krietsch
,
J.
,
Sanchez
,
G.
,
Inman
,
M.
,
Tsang
,
H.
,
Warr
,
M.
,
Passegué
,
E.
, et al. 
(
2015
).
Progressive chromatin condensation and H3K9 methylation regulate the differentiation of embryonic and hematopoietic stem cells
.
Stem Cell Rep.
5
,
728
-
740
.
Wei
,
C.
,
Zeff
,
R.
and
Goldschneider
,
I.
(
2000
).
Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7Rα, terminal deoxynucleotidyltransferase, and cμ expression
.
J. Immunol.
164
,
1961
-
1970
.
Welner
,
R. S.
,
Esplin
,
B. L.
,
Garrett
,
K. P.
,
Pelayo
,
R.
,
Luche
,
H.
,
Fehling
,
H. J.
and
Kincade
,
P. W.
(
2009
).
Asynchronous RAG-1 expression during B lymphopoiesis
.
J. Immunol.
183
,
7768
-
7777
.
Yona
,
S.
,
Kim
,
K.-W.
,
Wolf
,
Y.
,
Mildner
,
A.
,
Varol
,
D.
,
Breker
,
M.
,
Strauss-Ayali
,
D.
,
Viukov
,
S.
,
Guilliams
,
M.
,
Misharin
,
A.
, et al. 
(
2013
).
Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis
.
Immunity
38
,
79
-
91
.
Yoshida
,
H.
,
Honda
,
K.
,
Shinkura
,
R.
,
Adachi
,
S.
,
Nishikawa
,
S.
,
Maki
,
K.
,
Ikuta
,
K.
and
Nishikawa
,
S.-I.
(
1999
).
IL-7 receptor α+ CD3(−) cells in the embryonic intestine induces the organizing center of Peyer's patches
.
Int. Immunol.
11
,
643
-
655
.

Competing interests

The authors declare no competing or financial interests.

Supplementary information