Origin and Functions of Tissue Macrophages

Macrophages are distributed in tissues throughout the body and contribute to both homeostasis and disease. Recently, it has become evident that most adult tissue macrophages originate during embryonic development and not from circulating monocytes. Each tissue has its own composition of embryonically derived and adult-derived macrophages, but it is unclear whether macrophages of distinct origins are functionally interchangeable or have unique roles at steady state. This new understanding also prompts reconsideration of the function of circulating monocytes. Classical Ly6chi monocytes patrol the extravascular space in resting organs, and Ly6clo nonclassical monocytes patrol the vasculature. Inflammation triggers monocytes to differentiate into macrophages, but whether resident and newly recruited macrophages possess similar functions during inflammation is unclear. Here, we define the tools used for identifying the complex origin of tissue macrophages and discuss the relative contributions of tissue niche versus ontological origin to the regulation of macrophage functions during steady state and inflammation. Macrophages are distributed in tissues throughout the body and contribute to both homeostasis and disease. Recently, it has become evident that most adult tissue macrophages originate during embryonic development and not from circulating monocytes. Each tissue has its own composition of embryonically derived and adult-derived macrophages, but it is unclear whether macrophages of distinct origins are functionally interchangeable or have unique roles at steady state. This new understanding also prompts reconsideration of the function of circulating monocytes. Classical Ly6chi monocytes patrol the extravascular space in resting organs, and Ly6clo nonclassical monocytes patrol the vasculature. Inflammation triggers monocytes to differentiate into macrophages, but whether resident and newly recruited macrophages possess similar functions during inflammation is unclear. Here, we define the tools used for identifying the complex origin of tissue macrophages and discuss the relative contributions of tissue niche versus ontological origin to the regulation of macrophage functions during steady state and inflammation. Macrophages were first discovered late in the 19th century by Ilya Metchnikoff and are evolutionary conserved phagocytes that evolved more than 500 million years ago (Cooper and Alder, 2006Cooper M.D. Alder M.N. The evolution of adaptive immune systems.Cell. 2006; 124: 815-822Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, Tauber, 2003Tauber A.I. Metchnikoff and the phagocytosis theory.Nat. Rev. Mol. Cell Biol. 2003; 4: 897-901Crossref PubMed Scopus (57) Google Scholar). Metchnikoff combined both evolutionary and ontological perspectives, which is particularly relevant in light of recent findings that have revolutionized the macrophage field. In the 1960s, van Furth proposed that all tissue macrophages originate from circulating adult blood monocytes, which has been the prevailing view for the last 40 years despite evidence that tissue macrophages are independent of circulating monocytes (van Furth and Cohn, 1968van Furth R. Cohn Z.A. The origin and kinetics of mononuclear phagocytes.J. Exp. Med. 1968; 128: 415-435Crossref PubMed Google Scholar, Volkman et al., 1983Volkman A. Chang N.C. Strausbauch P.H. Morahan P.S. Differential effects of chronic monocyte depletion on macrophage populations.Lab. Invest. 1983; 49: 291-298PubMed Google Scholar, Sawyer et al., 1982Sawyer R.T. Strausbauch P.H. Volkman A. Resident macrophage proliferation in mice depleted of blood monocytes by strontium-89.Lab. Invest. 1982; 46: 165-170PubMed Google Scholar). However, in the last few years, a series of more definitive publications have drastically revised our understanding of macrophage origin by demonstrating that many resident tissue macrophages are in fact established during embryonic development and persist into adulthood independently of blood monocyte input in the steady state (Ginhoux et al., 2010Ginhoux 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.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (437) Google Scholar, Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (221) Google Scholar, Yona et al., 2013Yona S. Kim K.W. Wolf Y. Mildner A. Varol D. Breker M. Strauss-Ayali D. Viukov S. Guilliams M. Misharin A. et al.Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.Immunity. 2013; 38: 79-91Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Hashimoto et al., 2013Hashimoto D. Chow A. Noizat C. Teo P. Beasley M.B. Leboeuf M. Becker C.D. See P. Price J. Lucas D. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Epelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Guilliams et al., 2013Guilliams M. De Kleer I. Henri S. Post S. Vanhoutte L. De Prijck S. Deswarte K. Malissen B. Hammad H. Lambrecht B.N. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF.J. Exp. Med. 2013; 210: 1977-1992Crossref PubMed Scopus (14) Google Scholar, Jakubzick et al., 2013Jakubzick C. Gautier E.L. Gibbings S.L. Sojka D.K. Schlitzer A. Johnson T.E. Ivanov S. Duan Q. Bala S. Condon T. et al.Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes.Immunity. 2013; 39: 599-610Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Metchnikoff defined the embryonic establishment of macrophages in the starfish, which has no formal vascular system. Accordingly, he argued for inflammation independent of blood vessels, countering Julius Cohnheim’s duly influential claims that “there is no inflammation without blood vessels.” Thus, in the 21st century, we have come full circle to these debates with the reality that inflammation, even in organisms anatomically more complex than starfish, centrally involves macrophages but can occur through mechanisms that are both dependent and independent of monocyte recruitment from the vasculature. Self-sustaining resident macrophages already present in the tissue make this possible. As our tools develop, we continue to refine our understanding of tissue macrophages and can now further delineate multiple distinct embryonically derived macrophage lineages. Several detailed review articles describing different aspects of macrophage ontogeny and function have been published (Sieweke and Allen, 2013Sieweke M.H. Allen J.E. Beyond stem cells: self-renewal of differentiated macrophages.Science. 2013; 342: 1242974Crossref PubMed Scopus (15) Google Scholar, Davies et al., 2013aDavies L.C. Jenkins S.J. Allen J.E. Taylor P.R. Tissue-resident macrophages.Nat. Immunol. 2013; 14: 986-995Crossref PubMed Scopus (31) Google Scholar, Wynn et al., 2013Wynn T.A. Chawla A. Pollard J.W. Macrophage biology in development, homeostasis and disease.Nature. 2013; 496: 445-455Crossref PubMed Scopus (111) Google Scholar). Although a novel concept in and of itself, the dichotomy between embryonically derived and adult-derived macrophage populations does not fully encapsulate the entire complexity and diversity found among tissue macrophages. Intriguingly, tissue macrophages of differing ontological origins coexist, and when assessed as a group, they perform specialized, organ-specific functions. The goal of this review is to highlight emerging questions in the field, such as to what extent macrophage ontogeny versus the tissue niche dictates a macrophage’s function. Are we missing critical insight by not parsing macrophage function on the basis of origin? To what extent is there redundancy between adult-monocyte-derived macrophages and different resident macrophage subsets? We will utilize this framework to discuss how subsets of tissue macrophages and circulating monocytes originate, how expansion of tissue macrophages is regulated when homeostasis is disrupted, and what functions can be ascribed to individual subsets and lineages. During early gestation (embryonic day 6.5 [E6.5]–E8.5), macrophages are first observed and expand in the extraembryonic yolk sac during what is termed primitive hematopoiesis (Samokhvalov, 2014Samokhvalov I.M. Deconvoluting the ontogeny of hematopoietic stem cells.Cell. Mol. Life Sci. 2014; 71: 957-978Crossref PubMed Google Scholar) (see Figure 1). At this stage in development, macrophages are the only “white blood cell” produced, because restricted progenitors in the yolk sac give rise only to macrophages and red blood cells. This limited hematopoietic cell diversity is reminiscent of the Drosophila immune system (Makhijani and Brückner, 2012Makhijani K. Brückner K. Of blood cells and the nervous system: hematopoiesis in the Drosophila larva.Fly (Austin). 2012; 6: 254-260Crossref PubMed Google Scholar) and indicative of the conserved origin of the yolk-sac-derived macrophage lineage. Subsequently (E8.5–E10.5), definitive hematopoietic stem cells (HSCs) emerge from the aorta-gonad-mesonephros and give rise to all immune lineages. Beginning at E10.5, HSCs migrate to the fetal liver, which then serves as the major hematopoietic organ during the remainder of embryonic development. Only in the perinatal period do traditional bone marrow HSCs become the primary site of hematopoiesis and produce the full complement of immune lineages (Orkin and Zon, 2008Orkin S.H. Zon L.I. Hematopoiesis: an evolving paradigm for stem cell biology.Cell. 2008; 132: 631-644Abstract Full Text Full Text PDF PubMed Scopus (578) Google Scholar). During embryonic development, transcription factor usage and surface-marker expression differ between yolk-sac-derived and definitive-HSC-derived macrophages. Definitive HSCs are completely dependent on the transcription factor MYB, whereas yolk-sac-derived progenitors develop independently of MYB (Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (221) Google Scholar). Furthermore, during embryogenesis, yolk-sac-derived macrophages have a characteristic CX3CR1hiF4/80hiCD11blo expression pattern (Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (221) Google Scholar, Yona et al., 2013Yona S. Kim K.W. Wolf Y. Mildner A. Varol D. Breker M. Strauss-Ayali D. Viukov S. Guilliams M. Misharin A. et al.Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.Immunity. 2013; 38: 79-91Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Hashimoto et al., 2013Hashimoto D. Chow A. Noizat C. Teo P. Beasley M.B. Leboeuf M. Becker C.D. See P. Price J. Lucas D. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Epelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Unfortunately, many adult resident tissue macrophages that originate during embryonic development alter the expression of cell-surface markers as the animal matures, hindering the ability to precisely track macrophage populations. Clear examples include embryonically established tissue macrophages that express CX3CR1 during development (lung and peritoneal macrophages and liver Kupffer cells) but lose expression after birth (Yona et al., 2013Yona S. Kim K.W. Wolf Y. Mildner A. Varol D. Breker M. Strauss-Ayali D. Viukov S. Guilliams M. Misharin A. et al.Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.Immunity. 2013; 38: 79-91Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Genetic fate-mapping techniques give us the ability to precisely identify and track different embryonic macrophage populations into adulthood, and when combined with parabiotic and adoptive-transplant studies, they give us the ability to discern the relationship between macrophages and circulating blood monocytes. Fate-mapping studies utilize the principle of genetic recombination to permanently label cells (and all subsequent progeny) on the basis of the recombination-induced expression of a reporter gene that is under the control of a constitutive promoter (typically Rosa26). Genetic techniques have evolved to include inducible systems, where temporally controlling recombination facilitates precise labeling of embryonic populations and tracking into adulthood. Below, we will discuss in detail experimental evidence that undergirds the recently revised view of macrophage origins and identify both the strengths and the limitations of each approach (see a summary of tools used for defining macrophage origin in Table 1).Table 1Tools Used for Defining Macrophage LineagesStrategyResultStrengthLimitationReferencesRunx1CreERidentifies yolk-sac-derived macrophagesis expressed very early during developmenthas a very limited window; results in incomplete labeling during developmentHoeffel et al., 2012Hoeffel G. Wang Y. Greter M. See P. Teo P. Malleret B. Leboeuf M. Low D. Oller G. Almeida F. et al.Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages.J. Exp. Med. 2012; 209: 1167-1181Crossref PubMed Scopus (71) Google Scholar, Ginhoux et al., 2010Ginhoux 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.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (437) Google ScholarCsf1rCreERidentifies yolk-sac-derived macrophagesis expressed very early during development; has a longer window than Runx1CreERresults in incomplete labeling during developmentEpelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (221) Google ScholarFlt3Credefines cells that pass through definitive hematopoiesis (HSCs)has strong reporter induction in circulating monocytes in adult animals; differentiates well between embryonic and adult macrophages; is excellent at following population dynamics during stressdoes not differentiate well between embryonic lineages (yolk sac versus fetal monocytes); might not label some HSC-derived macrophages during times of rapid expansion (i.e., in early development or after adoptive transplant)Epelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (221) Google Scholar, Hashimoto et al., 2013Hashimoto D. Chow A. Noizat C. Teo P. Beasley M.B. Leboeuf M. Becker C.D. See P. Price J. Lucas D. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Boyer et al., 2011Boyer S.W. Schroeder A.V. Smith-Berdan S. Forsberg E.C. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.Cell Stem Cell. 2011; 9: 64-73Abstract Full Text Full Text PDF PubMed Scopus (23) Google ScholarCx3cr1GFP/+identifies cells currently expressing CX3CR1CX3CR1hi macrophages correspond well to yolk-sac-derived F4/80hiCD11blo macrophages during development onlyis not reliable for differentiating lineages in the adultYona et al., 2013Yona S. Kim K.W. Wolf Y. Mildner A. Varol D. Breker M. Strauss-Ayali D. Viukov S. Guilliams M. Misharin A. et al.Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.Immunity. 2013; 38: 79-91Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Jung et al., 2000Jung S. Aliberti J. Graemmel P. Sunshine M.J. Kreutzberg G.W. Sher A. Littman D.R. Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion.Mol. Cell. Biol. 2000; 20: 4106-4114Crossref PubMed Scopus (660) Google ScholarCx3cr1Crelabels all cells that pass through a CX3CR1+ stageidentifies traditionally CX3CR1− populations as having passed through the CX3CR1+ stageis less useful for differentiating between recruited and resident macrophages in the adultYona et al., 2013Yona S. Kim K.W. Wolf Y. Mildner A. Varol D. Breker M. Strauss-Ayali D. Viukov S. Guilliams M. Misharin A. et al.Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.Immunity. 2013; 38: 79-91Abstract Full Text Full Text PDF PubMed Scopus (108) Google ScholarCx3cr1CreERhas the potential to label embryonic macrophages at different stages of developmentidentifies very strong reporter expression in tissue macrophages expressing high levels of CX3CR1; only transiently labels monocytes, which allows for differential targetingis limited to tissue macrophages that express CX3CR1 at the time of inductionYona et al., 2013Yona S. Kim K.W. Wolf Y. Mildner A. Varol D. Breker M. Strauss-Ayali D. Viukov S. Guilliams M. Misharin A. et al.Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.Immunity. 2013; 38: 79-91Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, Goldmann et al., 2013Goldmann T. Wieghofer P. Müller P.F. Wolf Y. Varol D. Yona S. Brendecke S.M. Kierdorf K. Staszewski O. Datta M. et al.A new type of microglia gene targeting shows TAK1 to be pivotal in CNS autoimmune inflammation.Nat. Neurosci. 2013; 16: 1618-1626Crossref PubMed Scopus (13) Google Scholar, Parkhurst et al., 2013Parkhurst C.N. Yang G. Ninan I. Savas J.N. Yates 3rd, J.R. Lafaille J.J. Hempstead B.L. Littman D.R. Gan W.B. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.Cell. 2013; 155: 1596-1609Abstract Full Text Full Text PDF PubMed Scopus (13) Google ScholarRag1Creidentifies lymphomyeloid progenitorsis the only technique for specifically labeling lymphomyeloid progenitorsit is unclear whether progeny persist into adulthoodBöiers et al., 2013Böiers C. Carrelha J. Lutteropp M. Luc S. Green J.C. Azzoni E. Woll P.S. Mead A.J. Hultquist A. Swiers G. et al.Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells.Cell Stem Cell. 2013; 13: 535-548Abstract Full Text Full Text PDF PubMed Scopus (3) Google ScholarParabiosisdemonstrates tissue-specific replacement of resident macrophages by chimeric monocytesdoes not use irradiation; turnover is dependent on the steady state rather than tissue injury; use of Ccr2−/− recipients increases chimerism to >90%typically demonstrates only 30% chimerism in wild-type mice; surgery might alter steady-state traffickingHashimoto et al., 2013Hashimoto D. Chow A. Noizat C. Teo P. Beasley M.B. Leboeuf M. Becker C.D. See P. Price J. Lucas D. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Epelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Ajami et al., 2011Ajami B. Bennett J.L. Krieger C. McNagny K.M. Rossi F.M. Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool.Nat. Neurosci. 2011; 14: 1142-1149Crossref PubMed Scopus (130) Google Scholar, Ajami et al., 2007Ajami B. Bennett J.L. Krieger C. Tetzlaff W. Rossi F.M. Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.Nat. Neurosci. 2007; 10: 1538-1543Crossref PubMed Scopus (292) Google ScholarSublethal irradiation and bone marrow transplantinduces chimerism in blood monocytes without eradicating resident macrophagesdemonstrates blood monocyte dependence of tissue macrophages over long periods of time; tissue shielding can minimize local radiation injuryradiation might alter steady dynamics (i.e., injured tissue recruits monocytes)Epelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar Open table in a new tab The inducible runt-related transcription factor 1 (Runx1CreER)-based system takes advantage of the early expression of Runx1 in the yolk sac at ∼E7.0, which occurs prior to the development of definitive HSCs (Samokhvalov et al., 2007Samokhvalov I.M. Samokhvalova N.I. Nishikawa S. Cell tracing shows the contribution of the yolk sac to adult haematopoiesis.Nature. 2007; 446: 1056-1061Crossref PubMed Scopus (178) Google Scholar). Labeling of E7.0 Runx1-expressing yolk-sac-derived macrophages demonstrates that they persist faithfully into adulthood as microglia. Skin Langerhans cells contain both yolk-sac-derived and fetal-monocyte-derived populations, but fetal-monocyte-derived macrophages have become the dominant lineage over time (Hoeffel et al., 2012Hoeffel G. Wang Y. Greter M. See P. Teo P. Malleret B. Leboeuf M. Low D. Oller G. Almeida F. et al.Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages.J. Exp. Med. 2012; 209: 1167-1181Crossref PubMed Scopus (71) Google Scholar, Ginhoux et al., 2010Ginhoux 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.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (437) Google Scholar). Whereas early labeling techniques (prior to E7.5) enable specific tracking of yolk-sac-derived macrophages (such as microglia), labeling between E8.0 and E10.5 leads to recombination in both yolk-sac-derived macrophages and early definitive HSCs, which migrate to the fetal liver and give rise to blood monocytes and lymphocytes (Ginhoux et al., 2010Ginhoux 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.Fate mapping analysis reveals that adult microglia derive from primitive macrophages.Science. 2010; 330: 841-845Crossref PubMed Scopus (437) Google Scholar, Samokhvalov et al., 2007Samokhvalov I.M. Samokhvalova N.I. Nishikawa S. Cell tracing shows the contribution of the yolk sac to adult haematopoiesis.Nature. 2007; 446: 1056-1061Crossref PubMed Scopus (178) Google Scholar). As a result, the ability of Runx1CreER to differentiate between tissue macrophages and blood monocytes during later developmental stages becomes obscured. The inducible Csf1rCreER has also been used for fate-mapping approaches. Initially, it was observed that E8.5-labeled (yolk-sac-derived macrophages) seed virtually all developing tissues in the embryo and show variable persistence into adulthood (Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (221) Google Scholar). Similar to Runx1CreER, Csf1rCreER used at E8.5 in a separate study achieved ∼30% labeling of yolk-sac-derived macrophages in the embryo and persistent labeling of microglia at the same rate in adults, confirming the near exclusive yolk sac origin of these cells (Epelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The only other adult organs that retained significant numbers of yolk-sac-derived macrophages that originated from E8.5 were the heart and liver, suggesting that the persistence of early yolk-sac-derived macrophages might be more restricted than currently appreciated. However, given that yolk sac progenitors migrate to the fetal liver at later stages of development, the possibility exists that these progenitors can continue to give rise to adult tissue macrophages. Unlike using Runx1CreER, using Csf1rCreER to label at E8.5 did not result in labeling of adult circulating blood monocytes, suggesting that a longer window for fate-mapping studies might exist. Two important caveats should be highlighted. First, unlike Runx1CreER, Csf1rCreER is a transgene and might not report native gene expression in all conditions. Second, the precision of any tamoxifen-inducible system that requires temporal activation is limited because of the half-life of tamoxifen. For example, labeling early yolk sac progenitors at E8.5 could be confounded by the persistence of tamoxifen in tissue and labeling at later developmental stages, which underscores the importance of monitoring recombination not only in tissue macrophages but also in later HSC-derived cells. Adult definitive HSCs transiently pass through a FLT3+ stage during differentiation into all lineages (myeloid, lymphoid, megakarocyte, and erythroid cells), which can be exploited genetically (Flt3Cre) for labeling adult-definitive-HSC-derived cells (Boyer et al., 2011Boyer S.W. Schroeder A.V. Smith-Berdan S. Forsberg E.C. All hematopoietic cells develop from hematopoietic stem cells through Flk2/Flt3-positive progenitor cells.Cell Stem Cell. 2011; 9: 64-73Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). What is clear is that Flt3-Cre+ blood monocytes do not replace Flt3-Cre− tissue macrophage populations, even over extended periods of time (Epelman et al., 2014Epelman 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.Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation.Immunity. 2014; 40: 91-104Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, Hashimoto et al., 2013Hashimoto D. Chow A. Noizat C. Teo P. Beasley M.B. Leboeuf M. Becker C.D. See P. Price J. Lucas D. et al.Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes.Immunity. 2013; 38: 792-804Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Schulz et al., 2012Schulz C. Gomez Perdiguero E. Chorro L. Szabo-Rogers H. Cagnard N. Kierdorf K. Prinz M. Wu B. Jacobsen S.E. Pollard J.W. et al.A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science. 2012; 336: 86-90Crossref PubMed Scopus (221) Google Scholar). Interpreting the ontological origin of Flt3-Cre− macrophages must be handled with care because it might be temptin