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Microglial subtypes: diversity within the microglial community

2019; Springer Nature; Volume: 38; Issue: 17 Linguagem: Inglês

10.15252/embj.2019101997

1460-2075

Vassilis Stratoulias, José L. Venero, Marie‐Ève Tremblay, Bertrand Joseph,

Neurological Disease Mechanisms and Treatments

Review2 August 2019Open Access Microglial subtypes: diversity within the microglial community Vassilis Stratoulias Vassilis Stratoulias orcid.org/0000-0002-9724-6589 Toxicology Unit, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Jose Luis Venero Jose Luis Venero Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Marie-Ève Tremblay Marie-Ève Tremblay Department of Molecular Medicine, Université Laval, Quebec, QC, Canada Axe Neurosciences, Centre de Recherche du CHU de Québec-Université Laval, Quebec, QC, Canada Search for more papers by this author Bertrand Joseph Corresponding Author Bertrand Joseph [email protected] orcid.org/0000-0001-5655-9979 Toxicology Unit, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Vassilis Stratoulias Vassilis Stratoulias orcid.org/0000-0002-9724-6589 Toxicology Unit, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Jose Luis Venero Jose Luis Venero Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain Search for more papers by this author Marie-Ève Tremblay Marie-Ève Tremblay Department of Molecular Medicine, Université Laval, Quebec, QC, Canada Axe Neurosciences, Centre de Recherche du CHU de Québec-Université Laval, Quebec, QC, Canada Search for more papers by this author Bertrand Joseph Corresponding Author Bertrand Joseph [email protected] orcid.org/0000-0001-5655-9979 Toxicology Unit, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Author Information Vassilis Stratoulias1, Jose Luis Venero2,3, Marie-Ève Tremblay4,5 and Bertrand Joseph *,1 1Toxicology Unit, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden 2Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain 3Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Sevilla, Spain 4Department of Molecular Medicine, Université Laval, Quebec, QC, Canada 5Axe Neurosciences, Centre de Recherche du CHU de Québec-Université Laval, Quebec, QC, Canada *Corresponding author. Tel: +46 703057405; E-mail: [email protected] The EMBO Journal (2019)38:e101997https://doi.org/10.15252/embj.2019101997 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Microglia are brain-resident macrophages forming the first active immune barrier in the central nervous system. They fulfill multiple functions across development and adulthood and under disease conditions. Current understanding revolves around microglia acquiring distinct phenotypes upon exposure to extrinsic cues in their environment. However, emerging evidence suggests that microglia display differences in their functions that are not exclusively driven by their milieu, rather by the unique properties these cells possess. This microglial intrinsic heterogeneity has been largely overlooked, favoring the prevailing view that microglia are a single-cell type endowed with spectacular plasticity, allowing them to acquire multiple phenotypes and thereby fulfill their numerous functions in health and disease. Here, we review the evidence that microglia might form a community of cells in which each member (or "subtype") displays intrinsic properties and performs unique functions. Distinctive features and functional implications of several microglial subtypes are considered, across contexts of health and disease. Finally, we suggest that microglial subtype categorization shall be based on function and we propose ways for studying them. Hence, we advocate that plasticity (reaction states) and diversity (subtypes) should both be considered when studying the multitasking microglia. Introduction Microglia were introduced to the scientific literature a century ago (Río-Hortega, 1919a,1919b,1919c; Fig 1). During normal physiological conditions, microglial cells with a ramified morphology are regularly distributed throughout the central nervous system (CNS; Río-Hortega, 1919b). Upon pathology, microglia transform their morphology and function, leading to propose a cascade of "reaction" from ramified to hypertrophic and ameboid phenotypes that still orients research today (Flanary et al, 2007; Graeber, 2010; Fig 1). With the recent advances in genetic tools allowing for fate mapping (Ginhoux et al, 2010), microglia are now considered to be tissue-resident macrophages of the CNS that arise exclusively from the embryonic yolk sac (Alliot et al, 1999; Schulz et al, 2012; Kierdorf et al, 2013; Perdiguero et al, 2015). Microglia colonize the murine CNS from embryonic day (E)9.5 (Tay et al, 2017c) and represent a self-maintaining and long-lived cell population that persists for months, if not the entire lifespan of the organism (Lawson et al, 1992; Ajami et al, 2007, 2011; Mildner et al, 2007; Askew et al, 2017; Füger et al, 2017; Réu et al, 2017; Tay et al, 2017b). Beyond microglia functioning as mediators of injury, inflammation, and neurodegeneration, several roles in the healthy brain have been identified at an exponential rate this past decade (Cartier et al, 2014; Tremblay et al, 2015; Fig 1). Figure 1. Historical overview of microglial subtype identificationAlthough microglial subtypes have originally been proposed by Rio-Hortega in the first report of microglia, it was only recently that this idea was revisited. Download figure Download PowerPoint Microglia exhibit widely differing functions depending on the stage of life, CNS region, and context of health or disease. Differences in microglial number, morphology, and gene expression were also reported between sexes (Schwarz et al, 2012; Crain et al, 2013; Lenz et al, 2013; Pimentel-Coelho et al, 2013; Butovsky et al, 2015; Dorfman et al, 2017; Hanamsagar et al, 2017; Krasemann et al, 2017). Adequate microglial functions are crucial for plasticity and behavioral adaptation to the environment (Salter & Stevens, 2017; Tay et al, 2017a). Throughout life, microglia contribute to neurogenesis, neuronal circuit shaping, vascular formation and remodeling, and maintenance of homeostasis (Tay et al, 2017c). During aging and in diseases, these cells may become reactive or impaired in their surveillance and phagocytosis (Streit, 2002; Koellhoffer et al, 2017; Spittau, 2017). Microglial contribution to diseases is associated with compromised physiological roles (e.g., in synaptic maintenance and plasticity; Tay et al, 2017a) and processes that are adaptive in the healthy brain, yet leading to cell death and tissue damage in pathological settings (e.g., excitotoxicity, oxidative stress, and inflammation; Weil et al, 2008). Microglial reaction can be triggered by any kind of insults or disturbances to the CNS. Persisting microglial reaction, associated often with proliferation, is involved in pathological conditions ranging from neurodevelopmental disorders, traumatic injuries, infectious diseases, tumors, and psychiatric disorders, to neurodegenerative diseases. Depending on the stage of the life, CNS region, and stressor or pathological insult at play, the microglial reaction process was shown to proceed differently and to result in sometimes contrasting outcomes (see Fig 2A for a classical schematic representation, depicting a ramified gray microglial cell surrounded by a palette of colorful microglia each representing a distinct reaction state). It is also now recognized that microglia display a wide range of reaction states, a tremendous shift from the M1/M2 classification still used a few years ago (Martinez & Gordon, 2014; Ransohoff, 2016). According to this view, the numerous functions of microglia would be fulfilled through their reaction toward multiple phenotypes, each associated with a distinct molecular signature (Crain et al, 2013; Hickman et al, 2013; Butovsky et al, 2014; Bennett et al, 2016; Grabert et al, 2016; Flowers et al, 2017; Galatro et al, 2017; Keren-Shaul et al, 2017; Krasemann et al, 2017; Hammond et al, 2018; Masuda et al, 2019). However, several pieces of evidence also indicate that different pools of microglia might each display distinct intrinsic properties that would be acquired during their maturation or function within the CNS. These subtypes would co-exist at steady state and undergo further modulation or phenotypic transformation in response to stimuli (Fig 2B). Indeed, beyond the view that microglia are a unique cell type in the CNS that adopts different phenotypes in response to different stimuli, we propose in this review article that microglia might constitute a community of cells in which different members display distinct properties, perform distinct physiological functions, and respond differently to stimuli (Fig 2C). We review the distinctive features of several putative microglial "subtypes", at structural, ultrastructural, and expression levels, as well as their functional implications across contexts of health and disease. Furthermore, we propose to categorize microglial subtypes based on functions, rather than molecular signatures and markers. Finally, we suggest that microglial subtype candidates should be validated using a methodological workflow that we recommend. Figure 2. Microglial reaction states(A) Currently, microglia are considered a homogenous cellular population (core of the circle in gray) that is extremely plastic. Depending on the brain homeostasis status at a given developmental stage or resulting from pathology, microglia respond invariably to assume a wide range of phenotypes as described in the literature. (B) In the updated version proposed here, microglia constitute a heterogeneous cell population having intrinsic properties and functional specializations. (C) Upon an environmental cue, each microglial subtype may respond or not to the stimulus, by expanding and/or changing its morphology and gene expression to assume a specific phenotype. Download figure Download PowerPoint Microglia: a community fulfilling the vast microglial functions What defines a cell subtype is subject of intense debate, and it is discussed in Box 1 Accumulating evidence indicates that microglia are not the naïve cell type that invariably responds identically to any possible type of stimuli by assuming a predetermined phenotype. In fact, from a historical perspective, the notion of microglial subtypes had already been proposed in 1919 by Rio-Hortega in his original description of microglia (Río-Hortega, 1919b; Fig 1). He noticed that some microglia that he named "satellite" microglia were found in close proximity to neuronal cell bodies. A century later, we propose that the satellite microglia, which are discussed below, might represent one of the playing cards in the deck of microglial subtypes (Fig 3). It is important to acknowledge that others, avant-garde scientists, have paved the way for the concept of microglial diversity (McCluskey & Lampson, 2000; Olah et al, 2011; Hanisch, 2013; Gertig & Hanisch, 2014). Box 1: How to define a "cell (sub)type" The answer to "how to define a cell subtype?" is probably to be found in the answer to a closely related question, "how to define a cell type?" Traditionally, a cell type is defined based on its host tissue, morphology, lineage, function, and molecular composition. However, the definition of this term remains subject to intense debate (Clevers et al, 2017). The advancement of unbiased technologies for single-cell transcriptome profiling, such as high throughput single-cell RNAseq and mass cytometry (or improved/related methods), has revealed remarkable heterogeneity among cells which were traditionally considered to be homogeneous. However, whereas this degree of transcriptome and proteome heterogeneity is sufficient for defining cell subtypes, or even cellular states, is also a topic of intense debate (Trapnell, 2015; Okawa et al, 2018). While single-cell RNAseq and mass cytometry allow to define molecularly distinct cell subpopulations, these approaches require to be complemented by the identification of the unique functions associated with these cell populations, in order to define those as cell (sub)types. Worth a notice, it is of importance not to confound cell subtypes with cellular states of reaction. The latter is referring to the different phenotypes and associated functions a cell type may acquire in response to various stimuli. A cell subtype should be defined by shared properties/characteristics within other cells within the cell type. Their unique intrinsic features and selective physiological functions should also be independent from their microenvironment. These two concepts are not mutually exclusive, as a cell subtype in response to a stimulus could react and acquire a new phenotype, i.e., reaction states, thus adding another level of complexity. Microglial subtypes must be defined in steady-state and unchallenged conditions by their intrinsic propertie(s) which translate into unique physiological function(s). Typically, the existing literature is the foundation of a research plan, which by definition is biased in respect to studies aimed at identifying a new cell type or subtype. This includes any work with markers, most importantly staining, sorting, and isolation of cells. Reverse genetic approaches can provide a more reliable tool for such studies, but still they have inherit technical limitations such as cell gating in flow cytometry and antibody unspecificity (Luo et al, 2013). On the other hand, unbiased technologies such as single-cell RNAseq, mass cytometry, and electron microscopy are useful tools, but still we should be aware of their limitation in terms of providing a static view of cellular dynamics. They however become useful when combined with two-photon in vivo imaging to provide insights into dynamics. Serendipitous identification is also an approach, but it is sporadic and by definition non-systematic. All of the above methodologies can contribute to the identification of new microglial subtypes. Considering the various putative subtypes that we have discussed in this review, a need for classifying microglial subtypes is evident. Deciphering whether their variations are instructed by the microenvironment or whether they result from intrinsic properties is of prime importance, using the following methodological workflow: Fate-mapping strategies allowing to visualize selectively different microglial subsets, for instance using non-invasive chronic two-photon in vivo imaging—could be performed longitudinally across development, adulthood, and aging, under steady-state as well as disease conditions—to determine the identity of putative microglial subtypes as microglial subsets or phenotypes. Microglia could be considered subtypes if their defining properties remain when these cells are examined longitudinally, under steady-state or disease conditions. They would however be considered phenotypes if instead they can transform one into another, notably in response to stimuli. The molecular determinants and physiological roles of the distinct subsets could then be studied using a combination of gene and protein expression analyses, as well as morphology, ultrastructure, and dynamic investigations. Figure 3. Putative microglial subtypes with unique specializationsEmerging data provide support to the existence of putative microglial subtypes endowed with unique genomic, spatial, morphological, and functional specializations. We anticipate that analyzing these subtypes thoroughly, with the methodological workflow proposed in Box 1, and using a similar methodology for newly discovered ones, will result in the identification of a number of different microglial subtypes with unique functional characteristics that could be targeted for disease prevention or treatment. Download figure Download PowerPoint Microglial regional heterogeneity at steady state Although microglia are ubiquitously scattered throughout the CNS, their distribution varies across regions, also between the white matter and gray matter (Lawson et al, 1990). Microglial morphology differs with the presence of neuronal cell bodies, dendrites and axons, myelinated axons, and blood vessels. Furthermore, microglia exhibit regional differences in self-renewal and turnover rates under normal physiological conditions and upon stimuli, such as lipopolysaccharide (LPS) challenge (Lawson et al, 1992; Ajami et al, 2007, 2011; Mildner et al, 2007; Askew et al, 2017; Füger et al, 2017; Réu et al, 2017; Tay et al, 2017b; Furube et al, 2018). The regional microenvironment has been shown to tightly determine microglial identity at the transcriptional level, in both mouse and human (Gosselin et al, 2014, 2017). Direct evidence for microglial regional variability notably comes from studies in which microglia were isolated from wild-type, unchallenged adult mice, according to brain area, and their transcriptome was determined based on panels of pre-selected microglial markers. In one study, the expression of CD11b, CD40, CD45, CD80, CD86, F4/80, TREM2b, CX3CR1, and CCR9 was compared among microglia isolated from different CNS regions of young adult mice (de Haas et al, 2008). Although all of these markers were expressed across the CNS, their protein expression varied significantly between areas. In a similar study performed in adult rats, the expression levels of known microglial markers also showed region-specific profiles (Doorn et al, 2015). Similar studies performed in mice that compared microglia isolated from different brain areas additionally showed regional heterogeneity in expression pattern throughout the lifespan (Butovsky et al, 2014; Grabert et al, 2016; De Biase et al, 2017; Masuda et al, 2019). Additionally, in an unbiased single-cell RNA sequencing (RNAseq) study, in which cerebral tissue and hippocampal tissue from unchallenged young adult mice were analyzed, 47 molecularly distinct cell subtypes were identified, including two belonging to the microglia (Zeisel et al, 2015). These findings raise the intriguing possibility that regional differences in terms of neuronal survival, activity, growth factor release, metabolism, as well as synaptic plasticity, myelination, vascular remodeling, blood–brain barrier properties, may require distinct microglial functions, thus driving the differentiation of distinct microglial subtypes during development or function within the CNS. These microglial subtypes could be a major contributing factor to the microglial regional heterogeneity. Recently, cerebellar microglia were shown to display a unique clearance ability, defined by their expression of numerous genes supporting the engulfment and catabolism of cells or cellular debris (Ayata et al, 2018). This cerebellar microglial "type" is reminiscent of developing microglia and disease-associated microglia (DAM) that will be discussed below. By contrast, microglia from the striatum display a homeostatic surveillance phenotype. This microglial differentiation in response to regional differences in the environment was shown to be driven by epigenetic mechanisms (Ayata et al, 2018). In particular, the suppression of clearance genes in striatal microglia is mediated by PRC2, which catalyzes the repressive chromatin modification histone H3 lysine 27 trimethylation (H3K27me3). The ablation of PRC2 in microglia also results in the emergence of clearance microglia even in the absence of dying neurons, among both the striatum and cerebral cortex. These aberrant clearance microglia induce impaired motor responses, decreased learning and memory, together with the development of anxiety and seizures in mice (Ayata et al, 2018). A recent study that characterized the diversity of CNS-associated macrophages (CAM) also identified three different subsets of CAM that expressed high levels of Mrc1, Ms4at, Pf4, Stab1, Cbr2, CD163, and Fcrls, and were associated with different CNS compartments: the leptomeninges, choroid plexus, and perivascular space (Jordão et al, 2019). Consequently, some of the regional microglial diversity described using these markers could also be partly accounted for by CAM diversity. Microglial subtypes as defined by differential gene expressions Differential gene expression is an established approach for defining distinct subpopulations of a cell type, for instance the different neuronal subtypes (e.g., GABAergic and glutamatergic) observed in the healthy brain. In various contexts, neighboring microglia were shown to display differences in gene expression at steady state. These observed differences between microglia could arise from local cues, including interactions with different subtypes of neurons (e.g., inhibitory and excitatory) and glial cells (astrocytes, oligodendrocytes, and progenitors), or slight differences in signaling thresholds. Similarly, differences in peripheral macrophage activation by LPS and viruses have been described, where only a subset of the population concomitantly displays a response (Ravasi et al, 2002). In addition, microglia may directly communicate with each other, which suggests that the recruitment of a specific microglial cell might lead to an inhibition of the neighboring microglia. Microglia were initially defined as occupying non-overlapping territories in the healthy brain, but this view is now changing, with improved staining methods showing direct contacts between processes and sometimes cell bodies from neighbor microglial cells (for example, see Milior et al, 2016). Furthermore, the possibility that differential marker expression among adjacent microglia results from differences in microglial exposure to previous challenges also has to be considered. For instance, it has been shown using non-invasive two-photon in vivo imaging that neighbor microglia respond differently to laser injury in the intact, unchallenged brain, leading to their processes converging or not toward the site of injury (Nimmerjahn et al, 2005; Paris et al, 2018). In addition, microglial cell bodies were recently shown to migrate in the cerebral cortex (Eyo et al, 2018) and cerebellum (Stowell et al, 2018) of healthy adult mice, which paints another layer of complexity. However, the existence of microglial subtypes, each endowed with intrinsic differences in gene expression, cannot be excluded and we argue that the topic deserves further investigation. Putative microglial subtypes are discussed below: Keratan sulfate proteoglycan (KSPG)-microglia A quarter of century ago, microglia were shown in the unchallenged adult rat brain to exhibit constitutive heterogeneity in their expression of KSPG (Bertolotto et al, 1993), visualized in situ using the 5D4 monoclonal antibody (Fig 3). KSPG is located in the extracellular matrix and on the cell surface. They are suggested to contribute to the control of cellular adhesion and axonal growth. In particular, 5D4-KSPG is expressed by a subpopulation of ramified microglia, contrary to ameboid microglia and peripheral macrophages (Bertolotto et al, 1993, 1998). Of note, 5D4-KSPG expression does not coincide with the expression of GFAP, NG2, or MAP2, which relate to other CNS cells. The expression of 5D4-KSPG in microglia differs significantly between strains of inbred rats (Jander & Stoll, 1996b). In mammals, a subpopulation of 5D4-KSPG-expressing microglia was also reported in the spinal cord and retina (Bertolotto et al, 1993, 1998; Jander & Stoll, 1996a; Jones & Tuszynski, 2002; Zhang et al, 2005; Foyez et al, 2015). The 5D4-KSPG-microglia exhibit a preferential regional distribution in the CNS. Indeed, whereas these cells are found in large numbers among the hippocampus, brainstem, and olfactory bulb (OB), only few of them are detected in the cerebellum and cerebral cortex (Bertolotto et al, 1993, 1998). This putative microglial subset is also observed in the neonatal rat brain (Bertolotto et al, 1998). It is of importance to mention that 5D4-KSPG-microglia were shown to co-exist with 5D4-KSPG-negative microglia in the same CNS regions (Jones & Tuszynski, 2002). Although these studies argue for the presence of two different subtypes, based on KSPG-reactivity, a systematic approach is required to confirm this possibility (Fig 4). Figure 4.Toolbox. Download figure Download PowerPoint Hox8b-microglia These microglial cells have a molecular signature that differentiates them from the canonical population, together with a unique spatial and temporal distribution (see Box 2 for distinct ontogeny of Hoxb8-microglia). Mice carrying the driver Hoxb8-Cre and the reporter ROSA26-YFP alleles were crossed to trace YFP-Hoxb8 expression. In the adult brain, the only cells showing YFP signal appeared to be microglia. YFP-positive microglia were found throughout the brain, especially in the cerebral cortex and OB (Chen et al, 2010; De et al, 2018; Fig 3). YFP-positive microglia, which represent 25–40% of the total microglial population in the adult brain, were also shown to co-exist with YFP-negative microglia (Chen et al, 2010; De et al, 2018; Nagarajan et al, 2018). Transcriptomic analyses comparing Hoxb8-positive and Hoxb8-negative microglia revealed that they are very similar at steady state, with only 21 genes differing significantly in expression between the two populations (De et al, 2018). Hoxb8-microglia express microglial signature genes, such as Tmem119, Sall1, Sall3, Gpr56, and Ms4a7, and genes associated with hematopoietic ontogeny including Clel12a, Klra2, and Lilra5 at similar levels compared with non-Hoxb8 canonical microglia (Bennett et al, 2018; De et al, 2018). Of note, neither of the two putative microglial subtypes was found to expresses Hoxb8 in the adult brain; instead, the lineage tracer approach revealed that Hoxb8 is expressed by microglial progenitors prior to CNS infiltration (De et al, 2018). Selective inactivation of Hoxb8 in the hematopoietic system was also sufficient to induce pathological grooming behavior, as observed in constitutive Hoxb8 mutant mice (Chen et al, 2010; Nagarajan et al, 2018). The strategy for gene deletion included the use of Tie2 Cre mice that affect all hematopoietic cells and endothelial cells (Chen et al, 2010). More cell-specific deletion of Hoxb8 within microglial cells is a prerequisite to determine their selective involvement in pathological grooming behavior. Box 2 (with associated illustration): Revisiting the microglial origin(s) An important question arising from the existence of microglial subtypes relates to their possible origin(s). Do microglial subtypes possess intrinsic differences prior to populating the CNS, or do they acquire their unique properties once they have assumed their regional distribution within the CNS parenchyma? Current literature states convincingly that microglia derive from the first wave of hematopoiesis from the embryonic yolk sac in mouse (Ginhoux et al, 2010; Hoeffel et al, 2015; Perdiguero et al, 2015; Sheng et al, 2015; Mass et al, 2016), where they follow a stepwise maturation program (Mass et al, 2016; Matcovitch-Natan et al, 2016), before populating the embryonic brain at E9.5 (Tay et al, 2017c). Based on the above literature, microglial subtypes should differentiate once they have assumed their regional distribution inside the CNS parenchyma (a). This hypothesis could explain microglial differences resulting from regional differences in microenvironments or from differences in local cues among the microenvironment such as microglial interactions with different neuronal subtypes (inhibitory, excitatory) and glial cells (astrocytes, oligodendrocytes and their progenitors), or slight differences in signaling thresholds, leading to the observed differences in adjacent microglia. The alternative hypothesis which is based on microglial cells exhibiting intrinsic differences prior to infiltrating the CNS cannot be excluded at this early stage of investigation, and should be tested (b and c). In support of the later hypothesis, Capecchi et al reported that Hoxb8-microglia-progenitors already exist in the yolk sac at E8.5 (De et al, 2018). Subsequently, these cells transit through the aorta-gonad-mesonephros and fetal liver, where they expand in number, prior to their entry into the brain at E12.5 (De et al, 2018) (c). On the same lines, microglial cells found in CSF1R−/− (Ginhoux et al, 2010; Erblich et al, 2011) and in IL2-Tgfb1;Tgfb1−/− (Keren-Shaul et al, 2017) transgenic mice are expected to exhibit intrinsic differences prior to infiltrating the brain parenchyma. Recently, it has been reported that at E14.5 two microglial subpopulations exist, based on Ms4a7 expression (Hammond et al, 2018). It would be of great interest to investigate the ontogeny of these two subpopulations. In zebrafish, two waves of microglial infiltration have been reported (Xu et al, 2015; Ferrero et al, 2018). Microglia of a yolk-sac-equivalent structure origin initially populate the embryonic brain. Subsequently, the microglial population is replenished by adult microglia that derive from a distinct tissue later during zebrafish development (d). This microglial div