CORRECTION

Correction: Two distinct ontogenies confer heterogeneityto mouse brain microglia (doi: 10.1242/dev.152306)Shrutokirti De, Donn Van Deren, Eric Peden, Matt Hockin, Anne Boulet, Simon Titen and Mario R. Capecchi

RNA-seq data associated with Development (2018) 145, dev152306 (doi: 10.1242/dev.152306) are now available in GEO under accessionnumber GSE124710.

The inserted Data availability section is shown below, and both the online full-text and PDF versions have been updated.

Data availabilityRNA-seq data have been deposited in GEO under accession number GSE124710.

The authors apologise to readers for this omission.

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RESEARCH ARTICLE

Two distinct ontogenies confer heterogeneity to mousebrain microgliaShrutokirti De1,2,*, Donn Van Deren1,3,*, Eric Peden1, Matt Hockin1, Anne Boulet1, Simon Titen1 andMario R. Capecchi1,2,‡

ABSTRACTHoxb8 mutant mice show compulsive behavior similar totrichotillomania, a human obsessive-compulsive-spectrum disorder.The only Hoxb8 lineage-labeled cells in the brains of mice aremicroglia, suggesting that defective Hoxb8 microglia caused thedisorder. What is the source of the Hoxb8 microglia? It has beenposited that all microglia progenitors arise at embryonic day (E) 7.5during yolk sac hematopoiesis, and colonize the brain at E9.5. Incontrast, we show the presence of two microglia subpopulations:canonical, non-Hoxb8 microglia and Hoxb8 microglia. Unlike non-Hoxb8microglia, Hoxb8microglia progenitors appear to be generatedduring the secondwave of yolk sac hematopoiesis, then detected in theaorto-gonad-mesonephros (AGM) and fetal liver, where they aregreatly expanded, prior to infiltrating the E12.5 brain. Further, wedemonstrate thatHoxb8 hematopoietic progenitor cells taken from fetalliver are competent to give rise to microglia in vivo. Although the twomicroglial subpopulations are very similar molecularly, and in theirresponse to brain injury and participation in synaptic pruning, theyshow distinct brain distributions which might contribute to pathologicalspecificity. Non-Hoxb8 microglia significantly outnumber Hoxb8microglia, but they cannot compensate for the loss of Hoxb8 functionin Hoxb8microglia, suggesting further crucial differences between thetwo subpopulations.

KEY WORDS: Tmem119, Microglia, Hoxb8 microglia,Non-Hoxb8 microglia, Microglia ontogeny, Yolk sac,AGM and fetal liver hematopoiesis, Fetal HSCs, OCD,Obsessive-compulsive-spectrum disorders, Trichotillomania

INTRODUCTIONMicroglia are recognized as crucial players in the shaping and fine-tuning of newly formed brain circuits. Failure of proper microgliafunction can lead to behavioral pathologies (Kettenmann et al.,2013; Li et al., 2012; Nimmerjahn et al., 2005; Prinz and Priller,2014; Schafer et al., 2013; Tremblay et al., 2011; Zhan et al., 2014).The loss of Hoxb8 function in a subpopulation of microglia appearscausative for compulsive grooming and a hair removal pathologythat resembles the obsessive-compulsive-spectrum disorder (OCD),trichotillomania (Chen et al., 2010). Such new behavioral roles

for microglia greatly extend our vision of how these brainsentinels contribute to maintenance of brain homeostasis, beyondtheir accepted roles as guardians of order through theirphagocytic activity.

Based on temporal lineage tracing analysis, it has been reportedthat all microglia progenitors originate from the first wave of yolksac hematopoiesis at embryonic day (E) 7.5, and then directlypopulate the developing brain at E9.5 (Ashwood et al., 2006;Ginhoux et al., 2010; Gomez-Perdiguero et al., 2015; Hoeffel et al.,2015; Kierdorf et al., 2013; Schulz et al., 2012; Sheng et al., 2015).The nature of the progenitors generated during the second wave ofyolk sac hematopoiesis is currently under debate. Data presented inGomez-Perdiguero et al. (2015) and McGrath et al. (2015) supportthe generation of yolk sac erythromyeloid progenitors (EMPs) atE8.5 that subsequently give rise to a broad range of self-sustaining,tissue-resident macrophages. On the other hand, Sheng et al. (2015),using c-KitMerCreMer cell fate mapping, suggest that the second waveof yolk sac hematopoiesis is characterized predominantly by astem cell antigen-1 (Sca-1; Ly6a – Mouse Genome Informatics)-expressing progenitor cell population in the AGM and fetal liver,designated fetal hematopoietic stem cells (f-HSCs), which give riseto tissue-resident macrophages, except for microglia. After theformation of the blood brain barrier (BBB), resident microgliahave little exchange with the peripheral immune system, andmicroglial homeostasis is maintained through a balance of self-renewal and apoptosis (Ginhoux et al., 2010; Mildner et al., 2007).

In the course of characterizing a pathological grooming behaviorin Hoxb8 mutant mice, we identified a new subset of microgliain the brain that is uniquely labeled by a Hoxb8 lineage reporter(Chen et al., 2010). The developmental source of this subpopulationwas not determined. Henceforth, we will designate the populationof microglia labeled by the Hoxb8 cell lineage marker as Hoxb8microglia, and the canonical microglia population as non-Hoxb8microglia. Because grooming behavior is controlled by the centralnervous system, and the only cells in the brain labeled by theHoxb8lineage reporter appeared to be microglia, we suggested thatdefective Hoxb8 microglia caused the associated pathology (Chenet al., 2010). Consistent with this interpretation, the pathologicalovergrooming behavioral deficit could be rescued by transplantationof normal bone marrow into lethally irradiated Hoxb8 mutant mice.Further, the pathology is recapitulated by conditionally restrictingthe Hoxb8 mutation to the hematopoietic lineage, but not, forexample, by restricting themutation toHoxb8-expressing interneurons(Chen et al., 2010).

Herein, we show that theHoxb8microglia progenitors appear to begenerated during the second wave of yolk sac hematopoiesis, and arenot detected in the developing brain until E12.5, long after the arrivalof non-Hoxb8 microglia. Either a subpopulation of non-Hoxb8microglia turn onHoxb8 expression in the developing brain at E12.5,orHoxb8microglia progenitors enter the brain later because they haveReceived 16 March 2017; Accepted 4 June 2018

1Department of Human Genetics, University of Utah School of Medicine, Salt LakeCity, UT 84112, USA. 2Interdepartmental Program in Molecular Biology, Universityof Utah School of Medicine, Salt Lake City, UT 84112, USA. 3InterdepartmentalProgram in Neuroscience, University of Utah School of Medicine, Salt Lake City,UT 84112, USA.*These authors contributed equally to this work

‡Author for correspondence (mario.capecchi@genetics.utah.edu)

M.R.C., 0000-0002-9591-4993

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transited, for example, through other hematopoietic tissues, such asthe para-aortic-splanchnopleural (P-Sp)/aorto-gonad-mesonephros(AGM) region and/or the fetal liver. No Hoxb8 RNA transcripts areever detected in either the developing or adult mouse brain usingeither quantitative reverse transcription polymerase chain reaction(qRT-PCR) or deep sequencing to identify such transcripts, refutingthe first hypothesis. Thus, our data strongly support the secondhypothesis that Hoxb8 microglia enter the brain at E12.5 from ahematopoietic source within the embryo proper. Further, celltransplantation experiments involving the injection of geneticallymarkedHoxb8microglia progenitor cells, derived from E12.5 fetallivers, into neonatal mouse brains, clearly demonstrate thatthe purified Hoxb8 microglia progenitor cells are capable ofgenerating mature parenchymal microglia in vivo, as defined bytheir morphology and expression of the microglia-specific gene,Tmem119 (Bennett et al., 2016).Direct comparison of RNA transcripts by deep sequencing shows

that adult Hoxb8 and non-Hoxb8 microglia subpopulations are verysimilar molecularly, with only 21 genes showing significantdifferential expression. Moreover, two recently described markersthat distinguish parenchymal microglia from other macrophages inthe periphery or brain, Tmem119 and Sall1 (Bennett et al., 2016;Buttgereit et al., 2016), are similarly expressed in the two microglialsubpopulations. Both subpopulations also share similarities in theirresponse to brain injury and participation in synaptic pruning. Thesetwo populations of microglia, however, exhibit distinct stereotypicspatial distributions in the adult mouse brain. Interestingly,Hoxb8 microglia, unlike non-Hoxb8 microglia, show their highestconcentration in the brain in regions previously defined byfunctional magnetic resonance imaging (fMRI) in humans as the‘OCD circuit’ (Graybiel and Rauch, 2000). The juxtaposition ofHoxb8 microglia within the ‘OCD neural circuit’ could provide amechanism for the specificity of the behavioral disorder associatedwith the Hoxb8 mutation. In summary, our data define Hoxb8microglia as a bona fide microglia subpopulation. Although opento other interpretations, the expression of Hoxb8 in the earlyhematopoietic progenitor population, as well as the kineticsof amplification and distribution of the Hoxb8 hematopoieticprogenitors within hematopoietic tissues, suggest that thispopulation of microglia progenitor cells appears to be born duringthe second wave of yolk sac hematopoiesis and then transits throughthe AGM and fetal liver, where these cells are greatly amplifiedprior to their infiltration into the brain at 12.5 days of gestation. Thisalternative developmental pathway and route of entry into thedeveloping brain could provide Hoxb8microglia with opportunitiesto acquire functional diversity relative to non-Hoxb8 microglia.

RESULTSHoxb8 microglia progenitors are born during the secondwave of yolk sac hematopoiesis and expanded in the AGMand fetal liver prior to colonizing the E12.5 brainThe Hoxb8 lineage marker labels a subpopulation of parenchymalmicroglia (Chen et al., 2010); however, their developmental originhas not been previously defined. To determine the origin ofHoxb8 microglia, we generated Cx3cr1GFP/+; Hoxb8IRES-Cre/+;ROSA26CAG-LSL-tdTomato/+ mice. In these mice, Cre is expressed inall cells that express Hoxb8, leading to the constitutive expressionof the red, tdTomato fluorescent protein (Fig. 1B,C). This markeris maintained in the cells that expressed Hoxb8 and their progeny,irrespective of continued Hoxb8 expression. In these triple-transgenic mice, all parenchymal microglia are labeled withgreen fluorescent protein (GFP) due to Cx3cr1GFP expression

(Fig. 1A), whereas only Hoxb8 microglia are co-labeled withtdTomato (Fig. 1D-F). Initially, we monitored the appearance ofGFP+ only and GFP+ tdTomato+ microglia in the brain, as a functionof developmental time. At early time points (between E9.5 andE11.5), only GFP+ non-Hoxb8 microglia were observed in theembryonic brain by confocal microscopy (Fig. 2A,B). GFP+

tdTomato+ double-positive microglia, were absent at these earlytime points, but are consistently detected in the brain at E12.5,although such cells are rare at this stage (Fig. 2A,C). From E14.5onward, GFP+ tdTomato+ double-positive microglia are readilyapparent in the brain (Fig. 2A,D). Hoxb8microglia comprise ∼25%of the total microglia population in the adult brain cortex (Fig. 2A).The delayed appearance of Hoxb8 microglia could be explained byeither of two hypotheses: (1) a subpopulation of non-Hoxb8microglia activates the expression of Hoxb8 in the developing brainstarting around E12.5, or (2) a separate population of microglialprogenitors, marked by expression of theHoxb8-tdTomato reporter,starts to infiltrate the brain at E12.5. To address these hypotheses, wesought to quantify the spatial-temporal expression pattern of Hoxb8using qRT-PCR in Hoxb8 microglia progenitors residing in thedistinct hematopoietic tissues.

The emergence of Hoxb8 microglia progenitors duringembryogenesis (appearance of tdTomato-labeled CD41+ c-Kithigh-or CD45+ c-Kithigh-expressing cells) was determined by fluorescence-activated cell sorting (FACS) analysis of yolk sac tissue from E7.5 toE10.0, in the P-Sp/AGM region from E9.5 to E11.5 and fetal liverfrom E10.0 to E18.5. CD41 (Itga2b) is commonly used as a markerfor early, less mature, hematopoietic progenitors, whereas CD45(Ptprc) marks more mature hematopoietic progenitors (Ferkowiczet al., 2003; Mikkola et al., 2003). Hoxb8 microglial progenitors(CD41+ c-Kithigh tdTomato+) were not detectable during the earlystages of yolk sac hematopoiesis at E7.5 (Fig. 3D). Only low levels oftdTomato-positive progenitors (∼5.0%) were apparent in the yolk sacbetween E8.5 and E10.0 (Fig. 3A,D). In the AGM, a significantfraction (>40%) of CD41+ c-Kithigh progenitor cells was labeled withthe Hoxb8 cell lineage marker tdTomato by E10.0 during the earlystages of P-Sp/AGM hematopoiesis (Fig. 3B,D). It is evident fromP-Sp/AGM cell-counting experiments, that the number of tdTomato+

cells is greatly and selectively amplified, relative to those presentin the yolk sac (see below). By E11.5, greater than 75% of theseprogenitor cells were tdTomato+ (Fig. 3D). On the other hand, atE10.0 in the fetal liver, we could barely detect CD41+ c-Kithigh

tdTomato+ progenitor cells, but by E11.5, greater than 38% of theseprogenitor cells were tdTomato+ (Fig. 3D). Again, by comparingnumbers of tdTomato+ cells in fetal liver to yolk sac or AGM, thenumber of Hoxb8 progenitor cells in fetal liver relative to even AGMwas further amplified. By E18.5, almost 100% of the CD41+

hematopoietic progenitor population in the fetal liver was tdTomato+

(Fig. 3C,D). Because many progenitor cells in the fetal liver begin toexpress CD45, we also quantified the CD45+ c-Kithigh tdTomato+

population. These results were comparable with those from cellsorting for CD41+ c-Kithigh and tdTomato+ except that CD45marks alarger pool of hematopoietic progenitor cells in the later stages of fetalliver development (Fig. S1). The temporal and quantitative analysis ofthe Hoxb8 lineage-labeled progenitors in the yolk sac, P-Sp/AGMregion and fetal liver suggests that this progenitor cell populationfollows a developmental pathway that originates during the secondwave of yolk sac hematopoiesis. Expansion of this population beginsin the AGM, followed by further selective expansion in the fetal liverprior to colonization of the brain. When we compared hematopoieticprogenitor cell expansion rates between the yolk sac, AGM and fetalliver during their respective optimal periods of progenitor production,

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we observed a 16.6-fold increase in AGM, and a 281-fold increase infetal liver, relative to yolk sac. This represents approximately four andeight cell doublings, respectively.Sca-1 is a marker commonly used to identify hematopoietic stem

cells (HSCs) and is expressed in multipotent cells derived from E8.5yolk sacs (Inlay et al., 2014). To test whether Hoxb8 progenitorsexpress Sca-1, we used FACS to count progenitor cells derived fromthe yolk sac, P-Sp/AGM region and fetal liver during embryonicdevelopment, by gating for CD45+ Sca-1+ c-Kithigh tdTomato+

(Fig. 3E). tdTomato+ labels a significant fraction of the Sca-1+

f-HSC population in the yolk sac, P-Sp/AGM region and fetal liver.By E12.5, a time when Hoxb8 microglia first appear in the brain,the percentage of tdTomato+ cells in the CD45+ Sca-1+ c-Kithigh

population in the fetal liver approaches 90% (Fig. 3E).The Runx1 transcription factor has been shown to label

microglia progenitors during early mouse embryonic development(Ginhoux et al., 2010). To determine the extent of overlap betweenthe Hoxb8 lineage-labeled cells and the progenitor populationlabeled by Runx1, we generated Runx1IRES-GFP/+; Hoxb8IRES-Cre/+;ROSA26CAG-LSL-tdTomato/+ compound mice and analyzed the yolksac at E9.5 for CD41+ c-Kithigh GFP+ tdTomato− and CD41+

c-Kithigh GFP+ tdTomato+ progenitor cells (Fig. S2). We found that100% of all CD41+ c-Kithigh cells in the yolk sac at E9.5 expressedGFP, confirming results published by Ginhoux et al. (2010). Thepercentage of the GFP+ hematopoietic progenitor cells also labeledby tdTomato was ∼8%.Because the Hoxb8 lineage contributes to the hematopoietic

progenitor cell population in the yolk sac, P-Sp/AGM region and

fetal liver, we next asked whether adult HSCs are also derived fromthe Hoxb8 lineage. To address this issue, bone marrow was isolatedfrom Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+ adult mice andFACS analyzed for HSCs by gating for Lin− Sca-1+ c-Kithigh (LSK)tdTomato+ cells. Virtually all LSK+ cells were tdTomato+ (Fig. 3F).These sorted cells were further analyzed for the presence of Hoxb8transcripts by qRT-PCR and none were detectable. As adult bonemarrow LSK+ cells do not express Hoxb8, the Hoxb8 lineagemarker, tdTomato,must have been activated at an earlier hematopoieticprogenitor state (i.e. in f-HSCs).

The Hoxb8IRES-Cre and ROSA26CAG-LSL-tdTomato alleles provide aconvenient and sensitive marking system for the Hoxb8 microglialineage, but they do not reflect real-time Hoxb8 gene expression.Once the cell lineage marker is expressed, it stays on in those cellsand all of their progeny. Further, because the system is dependent onthe production and function of Cre protein, visualization of thereporter gene takes ∼24 h (i.e. for the synthesis of Cre protein,translocation to the nucleus, excision of the lox-stop-lox cassette, aswell as production of the reporter gene product).

In order to determine more accurately when and where Hoxb8transcripts are produced, we used qRT-PCR to analyze the yolk sac,AGM, fetal liver and brain in developing mouse embryos.Interestingly, Hoxb8 transcripts were most abundant in the yolksac at E8.5 (Fig. 4). Although expression ofHoxb8was detectable inthe AGM and fetal liver, this expression was significantly less thanin the yolk sac at E8.5. In stark contrast, we did not detect anyHoxb8transcripts at any time point analyzed in the head region ofdeveloping embryos from E9.5 to birth (Fig. 4). In addition, by

Fig. 1. Two-color microglia mouse model. (A-F) All microglia in the brain are labeled with the fractalkine receptor (Cx3cr1) disrupted by the GFP gene(A,D) (Jung et al., 2000), and Hoxb8microglia are also labeled by the Hoxb8 cell lineage marker Hoxb8IRES-Cre, ROSA26CAG-LSL-tdTomato (B,C,E) (Madisen et al.,2010). (F) Note that only a subpopulation of microglia is co-labeled with the Hoxb8 cell lineage marker. The above confocal micrographs depict an imagefrom the frontal cortex of an adult mouse. Scale bars: 30 µm.

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RNA deep sequencing, Hoxb8 transcripts could not be detectedin mature Hoxb8 microglia present in the adult brain parenchyma(see below). Thus, Hoxb8 is predominantly expressed in asubpopulation of yolk sac hematopoietic progenitor cells during avery narrow window of Hoxb8 microglia progenitor development,long before their infiltration into the brain. Taken together, (1) thegene expression pattern of Hoxb8 in the yolk sac, AGM and fetalliver, (2) the lack of expression of Hoxb8 in the developing andmature brain, (3) the distribution pattern of tdTomato-expressingprogenitors from the yolk sac, to AGM and fetal liver, and (4) thetime of entry ofHoxb8-labeled progenitor cells into the developingbrain, we concluded that Hoxb8 microglia have a significantlydifferent embryonic history, relative to non-Hoxb8 microglia.In summary,Hoxb8microglia progenitors appear to be born during

the second wave of yolk sac hematopoiesis. Sheng et al. (2015) hassuggested that the progenitor population coincident with the secondwave of hematopoiesis is characterized by expression of Sca-1.Consistent with this interpretation, most Hoxb8 hematopoieticprogenitor cells express Sca-1, which is not known to be expressedduring the first wave of yolk sac hematopoiesis. TheHoxb8 progenitorpopulation then appears in the AGM and fetal liver, where Hoxb8lineage cells extensively and selectively expand in number (which isalso characteristic of the secondwave of yolk sac hematopoiesis), priorto their infiltration into the developing brain beginning at E12.5. Theadded exposure ofHoxb8microglia progenitor cells to the AGM andfetal liver prior to their entry into the brain provides opportunities forsignaling inputs from these hematopoietic organs, and therebypotential acquisition of additional genetic and epigenetic complexitythat might be translated into a new repertoire of functions.

Early postnatal dynamics of Hoxb8 microgliaHaving shown that Hoxb8 microglia have a distinct ontogenycompared to canonical microglia, we next set out to determinewhether the distinct embryonic history affected Hoxb8 microgliabiology. Hoxb8 microglia cell distribution and morphology changeconsiderably during early postnatal development (Fig. S3), consistentwith previously reported observations on early postnatal developmentof canonical microglia (Dalmau et al., 2003).

The proportion of Hoxb8 microglia compared to non-Hoxb8microglia increases several fold during the first postnatal weekand remains relatively constant at ∼20-25% thereafter (Fig. 2A).Following BBB closure, infiltration of microglia should be severelylimited and the proportion of the two microglia populations shouldremain constant throughout development. We therefore soughtto determine whether the relative enrichment of postnatalHoxb8 microglia was due to higher proliferation rates. Usingincorporation of 5-ethynyl-2´-deoxyuridine (EdU) into dividingcells, we found that the overall proliferation in cortical microgliawas high during the first postnatal week but decreased to barelymeasurable values by postnatal day (P) 16 (Fig. S4A). Hoxb8microglia show a higher proliferation rate just after birth (Fig. S4A).Beyond P4, proliferation in the two subpopulations did not appear todiffer significantly. To complement the proliferation assays with ameasurement of cell death, we evaluated microglia for the presenceof activated caspase-3, a primary effector of apoptosis. Microglialapoptotic rates were observed to be relatively high at P0 (∼1.5%)(Fig. S4B), but decreased dramatically by P4 and remained at nearlyundetectable levels thereafter. No significant differences in celldeath were detected between the two subpopulations of microglia.

Fig. 2. Hoxb8 microglia enter the brainsignificantly later than non-Hoxb8microglia. (A) Graph showing the Hoxb8microglial population (orange) as apercentage of total microglia in thedeveloping mouse brain cortex (E10.5-P16)as determined by confocal microscopy.P-values were determined fromcomparisons with the previous time point.(B-D) Hoxb8 (yellow, white asterisks) andnon-Hoxb8 (green) microglia in E10.5 (B),E12.5 (C) and E14.5 (D) brain parenchyma.Scale bars: 50 µm. n=3 biological replicatesper time point, ns, non-significant; *P<0.05,**P<0.005, ***P<0.0001; data aremean±s.e.m. See also Figs S3 and S4.

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Gene expression profile of Hoxb8 microgliaNext, we compared the gene expression profiles of non-Hoxb8microglia and Hoxb8 microglia. Microglia were isolated from12-week-old animals carrying the Cx3cr1GFP/+; Hoxb8IRES-Cre/+;ROSA26CAG-LSL-tdTomato/+ alleles. To reduce potential contaminationfrom peripheral monocytes, the brains were perfused and themeninges removed. Additionally, microglia were isolated fromforebrain and midbrain tissue to eliminate potential contaminationresulting from tdTomato-labeled interneuron projections emanatingfrom the spinal cord (Fig. 8D). Microglia were sorted, gating ontdTomato and GFP. The barcoded complementary DNA (cDNA)libraries were sequenced on Illumina HiSeq platforms, resulting in25-50 million sequences per biological sample. The data sets werevery similar with only 21 genes showing differential expressionlevels between the non-Hoxb8 microglia and Hoxb8 microglia

subpopulations (Table S1). Fig. 5 provides a summary of the RNAsequence data, illustrating the fold difference in gene expression(relative to expression in non-Hoxb8microglia) for all genes with aquantifiable expression level. Each point on the plot represents asingle gene as identified in the GRCm38v3 mouse genomicassembly. The size of the point is proportional to the statisticalsignificance of the observed fold change values for each gene. Thelargest symbols denote genes with the lowest adjusted P-valuesfrom our DEseq2 analysis. The color of the dot denotes relativeranking in this set (see Fig. 5 legend).

The list of significantly differentially expressed genes isremarkably small (Table S1). It is an interesting list of genes, notedfor their expression in myeloid cells representing a broad range ofactivities: receptors participating in cell signaling pathways, ligandssuch as cytokines and chemokines, activators of cell mobility,

Fig. 3. Hoxb8 microglia progenitors are selectively expanded during AGM and fetal liver hematopoiesis. (A) FACS profiles of CD41+ cells gated forc-Kit and tdTomato from the yolk sac of E8.5 and E9.5 Cx3cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+ embryos. (B) FACS profile of CD41+ cells gatedfor c-Kit and tdTomato isolated from the AGM of E10.0 and E11.5 embryos. (C) FACS profiles of CD45+ cells gated for c-Kit and tdTomato from fetal livers ofE14.5 and E18.5 Cx3cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+ embryos. (D) Graph showing the percentage of Hoxb8 hematopoietic progenitorcells (CD41+ c-Kithigh tdTomato+) through embryonic development in different tissues. n=2-4 replicates or pooled replicates per time point; data are mean±s.e.m.See also Fig. S1. (E) Graph showing the percentage of Hoxb8 f-HSCs (CD45+ Sca-1+ c-Kithigh tdTomato+) through development in different tissue. n=2-6replicates or pooled replicates per time point; data are mean±s.e.m. (F) Graph depicting the percentage ofHoxb8HSCs (Lin− Sca-1+ c-Kithigh tdTomato+) in adultbone marrow. n=5 replicates; data are mean±s.e.m. See also Figs S1 and S2.

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protein kinases, inhibitors of tyrosine kinase signaling, and proteinsassociated with inflammatory responses. Although this is aprovocative list, what is strikingly clear is that these two microgliasubpopulations share highly similar expression profiles.When we looked at the level of Hoxb8 expression, the number of

Hoxb8 transcripts detected in adult Hoxb8 microglia is well belowone transcript per cell. This observation, coupled with the qRT-PCRdata (Fig. 4), shows that Hoxb8 expression in embryonic and adultbrains is not detectable, indicating that the Hoxb8 cell lineage

marker (tdTomato) is activated in Hoxb8 microglia progenitor cellswell before their infiltration into the brain.

Recently, the Barres laboratory described a marker expressed inmature microglia, Tmem119, which distinguishes parenchymalmicroglia from other macrophages in the brain and periphery(Bennett et al., 2016). In our comparative mouse transcript analysisof non-Hoxb8 and Hoxb8 microglia, we found that Tmem119was significantly and similarly expressed in both microglialsubpopulations (10,940 and 11,765 reads, respectively; Table S2).

Fig. 4. Hoxb8 expression through embryonic development. Hoxb8 expression is predominantly detected in the yolk sac during primitive hematopoiesis asdetermined by qRT-PCR in the respective tissues as a function of developmental time. n=3-5 replicates per time point; data are mean±s.e.m. Data werenormalized to GAPDH expression.

Fig. 5. Few genes are differentially expressed between Hoxb8 and non-Hoxb8 microglia. A representation of differentially expressed genes with quantifiableexpression levels plotted, comparing Hoxb8 microglia with non-Hoxb8 microglia. Note the linear scale on the y-axis (fold change expression). Each point onthe plot represents a single gene as identified in the GRCm38v3 mouse genomic assembly. Plot symbol size is proportional to the statistical significance ofthe observed fold change values for each gene. The largest symbols denote genes with the lowest adjusted P-values (padj) from our DEseq2 analysis. Genesconsidered statistically significant according to the default DESeq2 guidelines (padj <0.1) are coded red; the color saturation indicates their relative ranking in thisset (padj <0.1). Thus, themost significantly differentially regulated gene is displayed as a fully saturated red circle, while the genewith the highest padj value still lessthan 0.1 is displayed as a faint red circle. Similarly, geneswithin padj values 0.1-0.2 are displayed as blue circles, while geneswith padj between 0.2-1.0 are displayedas gray circles. For actual fold differences of the top differentially expressed genes, see Table S1 and padj values for all genes provided in Table S2.

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The presence of the Tmem119 marker in both subpopulationsfurther supports our classification of Hoxb8 microglia asparenchymal microglia.Sall1 has also been described as a marker whose expression is

largely restricted to microglia and has not been detected in otherperipheral or brain macrophages (Buttgereit et al., 2016). Notably,this marker is also expressed at equivalent levels in the twomicroglial subpopulations (9500 versus 9116 reads in non-Hoxb8and Hoxb8 microglia, respectively; Table S2).The Hoxb8 lineage reporter system, Hoxb8IRES-Cre;

ROSA26CAG-LSL-tdTomato also labels peripheral macrophages.The question then arises: how many of the tdTomato+ cells in thebrain are legitimate parenchymal microglia as opposed to othermyeloid cells present in the normal brain? To answer this questionwe used an anti-Tmem119 antibody to quantify the percentage oftdTomato+ cells that are specifically microglia. Examination ofbrain sections derived from adult mice (n=3) counterstained with theanti-Tmem119 antibody (Fig. S5) and quantified for co-labeledcells revealed that greater than 98% of the tdTomato+ cells in thesesections were parenchymal Hoxb8 microglia and greater than 99%of the GFP only cells were parenchymal non-Hoxb8 microglia.

Hoxb8 hematopoietic progenitor cells derived from E12.5fetal liver give rise to mature parenchymal microglia whenintroduced into neonatal mouse brainsHaving shown that Hoxb8 microglia in adult mice are labeledwith the microglia-specific marker, Tmem119, we were positionedto directly determine whether fetal liver derived Hoxb8-labeledc-Kithigh, hematopoietic progenitor cells, are capable of giving riseto mature parenchymal microglia when transplanted into neonatalmouse brains. Freshly sorted hematopoietic progenitor cells(tdTomato+ c-Kithigh) derived from E12.5 fetal livers ofCx3cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+ mice wereinjected into the brains of C57Bl6/J pups at P4, whichwere allowed todevelop to P14-P17 to provide time for the expression of the mature

microglial-specific marker, Tmem119. Brain sections of these micewere stained with anti-tdTomato, anti-GFP and anti-Tmem119antibodies, and scanned by confocal microscopy. Representativeimages are shown in Fig. 6. Transplantation of 25,000 c-kithigh

tdTomato+ hematopoietic progenitor cells into C57Bl6/Jpups resulted in low engraftment of GFP+ tdTomato+ cells(∼50 cells/brain), all of which exhibited the morphology of maturemicroglia and co-labeled with Tmem119. Doubling the numberof transplanted progenitor cells resulted in higher engraftment(∼350 cells/brain). However, some of these cells appearedmorphologically less mature and 83% of these cells co-labeledwith Tmem119. Thus, overabundance of transplanted cells appearsto slow down the maturation of the engrafted cells. As expected,under both experimental conditions, all tdTomato+ cells alsoexpressed GFP resulting from the presence of the Cx3cr1GFP

allele in the donor cells. Finally, the engraftment efficiency of thefetal liver-derived Hoxb8 progenitor cells was enormouslyincreased by using Csfr1−/− mutant pups as recipients in order toablate the resident microglia population in the recipients prior totransplantation (Fig. S6) (Erblich et al., 2011). The recipient Csf1r−/−

brain was engrafted with thousands of tdTomato+ GFP+ Hoxb8microglia cells. The latter experiments suggest that most of the newlyintroduced Hoxb8 progenitor cells in the previous experiments wereeliminated from the recipient brains by the resident brain microglia inthe recipient wild-type brain. Because the only source of tdTomato+

GFP+ cells in these pups were donor-derived, purified hematopoieticprogenitor cells obtained from the fetal livers of E12.5 embryodonors, we conclude that these progenitor cells are indeed capable ofgiving rise to mature parenchymal microglia when introduced intowild-type and Csf1r−/− mutant mouse brains.

Physiological properties of Hoxb8 microgliaThe activation response of microglia following an insult to braintissue is one of the most well-characterized microglial functions. Weanalyzed acute and chronic injury responses of the two microglial

Fig. 6. E12.5 Hoxb8 fetal liver-derived hematopoietic progenitors give rise to parenchymal microglia in vivo. Mature GFP+ tdTomato+ Tmem119+

microglia, generated from transplanted Hoxb8 lineage progenitor cells, are marked by white asterisks. Tmem119 immunohistochemistry identifies both donor-derived GFP+ tdTomato+ microglia and recipient resident microglia (no fluorescence). Scale bars: 20 µm. See also Fig. S6.

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subpopulations in the cortex of the adult mouse brain to comparetheir effectiveness as brain macrophages.To study the acute response to injury, we employed multiphoton

microscopy for live imaging in the mouse brain cortex. For thispurpose, theCx3cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+

triple-transgenicmouse linewas used to visualize both subpopulationsof microglia simultaneously (Fig. 7A-D). The response of activatedmicroglia to focal laser ablation in the brain cortex was observed inanesthetized mice (Movie 1). Immediately following laser-inducedablation, the microglial processes started to advance toward thedamage, and within 20-25 min they reached the site of injury(Fig. 7A-D, Movie 1). Both classes of microglia responded withsimilar speed (Hoxb8 microglia, 1.34±0.19 µm/min; non-Hoxb8microglia, 1.49±0.4 µm/min), showing that both subpopulationshave indistinguishable acute responses.To analyze the response to chronic injury, adult mouse brains

were subjected to a small, localized needle poke via intracranialinjection, followed by a recovery period of 7 days. This recoveryperiod provides time for microglia surrounding the injection site tobe activated and migrate to the site of injury. Hoxb8 microgliademonstrated a greater tendency to accumulate around the site of

injection (Fig. 7E,F). These cells exhibited a 1.9-fold increase in celldensity around the injury site compared with a 1.1-fold increase inthe density of non-Hoxb8 microglia (Fig. 7E,F). Because thismechanical injury created some damage to the surroundingvasculature, it might also have led to the infiltration of tdTomato-expressingHoxb8 lineage macrophages present in peripheral blood.To distinguish the resident Hoxb8 microglia from peripheralmacrophages, we stained the cells with the microglia-specific 4D4antibody (Dr Oleg Butovsky, Harvard Medical School, Boston,MA, USA) (Fig. S7). The 4D4 antibody labels resident brainmicroglia specifically in both the resting and activated states. Thisanalysis showed that the majority of Hoxb8 lineage cells (>93%)around the site of injury were resident brain Hoxb8microglia with asmall contribution from peripheral cells. These results suggest thatHoxb8 microglia are more responsive than non-Hoxb8 microglia tochronic injury in the brain.

Synaptic pruningEarly in the normal development of the brain, far more synapticconnections are formed than are maintained. Activity-dependentsynaptic pruning is used to eliminate weaker connections and thereby

Fig. 7. Hoxb8 microglia have similarresponse to damage as non-Hoxb8microglia. (A-D) Stills from a time-lapsemovie of microglial response to damageinduced by focused laser ablation in braincortex, using in vivo multiphoton imaging.Scale bars: 30 μm. (A) Hoxb8 (yellow) andnon-Hoxb8 (green) microglia in their restingstate before injury. A video of the acuteresponse is provided (Movie 1).(B-D) Successive time points as themicroglial processes converge towards thesite of injury. (E)Microglial activation inducedby needle-poke injury (white dashed lines)in the mouse brain cortex observed 7 dayspostinjury. Scale bars: 50 μm. (F) Hoxb8microglia show a 1.9-fold increase innumbers around the injury site comparedwith a 1.1-fold increase in non-Hoxb8microglia. n=5 mice, ns, non-significant;*P<0.05; data are mean±s.e.m. See alsoFig. S7 and Movie 1.

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maintain strengthened synapses (Hua and Smith, 2004; Katz andShatz, 1996; Sanes and Lichtman, 1999). Microglia have been shownto actively participate in this synaptic remodeling program (Paolicelliet al., 2011; Schafer et al., 2012, 2013; Stephan et al., 2012). Aparticularly attractive system to assess the role ofmicroglia in activity-dependent synaptic pruning is the reduction of excess synapses in themouse retinogeniculate nucleus (Schafer et al., 2012; Stephan et al.,2012). These neurons can be labeled through ocular injection ofanterograde tracers, such as the cholera toxin β subunit conjugated toAlexa Fluor 647 (CTB-A647) (Schafer et al., 2012). By carrying outthese experiments in our triple-transgenic mice, we directly comparedthe efficiency of non-Hoxb8 microglia and Hoxb8 microglia tophagocytose labeled neurons. The two microglia populations wereequally competent in mediating activity-dependent synaptic pruning

in the retinogeniculate nucleus during its peak activity in P5mice (Fig. 8A-C).

Distribution of Hoxb8 microglia in the adult mouse brainDuring embryogenesis and early postnatal development, weobserved higher concentrations of Hoxb8 microglia in the cortexthan in other neighboring regions of the brain. To determinewhether this bias persisted into adulthood, we examined thedistribution of the two microglial subpopulations in the adult brain.In Fig. 8D, the strong tdTomato signal in the posterior brain (rightside) is associated with ascending fibers emanating from the spinalcord, such as the dorsal and ventral spinocerebellar tracts (DSC andVSC), and the spinoreticular tracts (SR). These fibers stronglyexpress tdTomato becauseHoxb8 is strongly expressed in the dorsal

Fig. 8. Synaptic pruning behavior of Hoxb8 andnon-Hoxb8 microglia is similar, whereas thedistributions of the two microglial populations in theadult brain are significantly different. (A) Confocalimage of Hoxb8 and non-Hoxb8 microglia, situatedclose to the CTB-A647-labeled neurons in the dLGN.(B) 3D rendering of the microglial cells shown in A.The yellow and green spots indicate the phagocytosedtracer puncta detected inside the Hoxb8 and non-Hoxb8microglia, respectively. Scale bars: 10 μm. (C) Histogramshowing the average number of neuronal pieces,phagocytosed detected per unit volume of a microglialcell. n=3 mice; ns, non-significant; **P<0.005; data aremean±s.e.m. (D) Spatial distribution of Hoxb8 microglia.Comparative distribution patterns of non-Hoxb8 microglia(green) and Hoxb8 microglia (yellow) in the adult mousebrain. LO, lateral orbital cortex; CPu, caudate putamen;DSC and VSC, dorsal and ventral spinocerebellar tracts;M1/2, primary and secondary motor cortex; VP, ventralpallidum; SNR, substantia nigra; SR, spinoreticular tracts;ST, spinothalamic track. The strong fluorescence in theposterior region of the brain reflects Hoxb8-expressinginterneuron projections emanating from the spinal cord.

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spinal cord interneurons that receive inputs from peripheral sensoryneurons (Holstege et al., 2008). Although these interneuronsexpress high levels of Hoxb8, selective disruption of Hoxb8 inthese interneurons using conditional mutagenesis did not recapitulatethe compulsive pathological overgrooming behavior characteristic ofHoxb8mutant mice (Chen et al., 2010), ruling out any contribution ofthese interneurons to the pathological overgrooming phenotype.In the adult brain parenchyma, the concentration of Hoxb8

microglia relative to non-Hoxb8 microglia was noticeably higher inthe cortex, specifically in the frontal association cortex, the primaryand secondary motor cortex (M1/2), and the lateral orbital cortex(LO). The dorsal striatum (caudate putamen, CPu) and the olfactorybulb also display relatively higher concentrations ofHoxb8microglia(Fig. 8D). In contrast, other regions such as the ventral pallidum(VP) and substantia nigra (SNR) show much lower densities ofHoxb8 microglia. Interestingly, the regions of high Hoxb8 microgliaconcentrations coincided with brain regions previously designated asthe ‘OCD circuit’, based on fMRI studies of OCD and control humanpopulations (Graybiel and Rauch, 2000). The relative distributionpatterns of non-Hoxb8 and Hoxb8 microglia in the adult mousebrain were reproducible from mouse to mouse. The distinct spatialdistribution of Hoxb8microglia in the normal brain might contributeto the specificity of behavioral phenotype observed in Hoxb8mutant mice.

DISCUSSIONA hallmark of the immune system is its diversity and complexity.Thus, it should not be surprising that diversity and complexity arealso characteristics of microglia, a cell type that is derived from theimmune system and is noted for performing multiple biologicalfunctions in the brain. Herein, we show that, in contrast topreviously published results, mammalian microglia have more thanone developmental route to colonize the embryonic brain. Thecanonical microglia progenitors, generated during the first wave ofyolk sac hematopoiesis at E7.5, transit directly from extraembryonichematopoietic tissue (i.e. yolk sac) to the developing brain. Althoughopen to alternative interpretations, parsimony and consistency withthe data derived from both Hoxb8 expression profiling as well asthe progressive locations of the Hoxb8 hematopoietic progenitorpopulation during development as shown by FACS analysis,suggest that Hoxb8 microglia progenitors are generated during thesecond wave of yolk sac hematopoiesis then seed the AGM andfetal liver, where they are greatly amplified prior to their infiltrationinto the brain at E12.5. Consistent with the above hypothesis,Hoxb8 microglia progenitors enter the brain significantly later(E12.5) than non-Hoxb8 microglia (E9.5) (Ginhoux et al., 2010;Hoeffel et al., 2015; Kierdorf et al., 2013; Schulz et al., 2012;Sheng et al., 2015). Furthermore, purified Hoxb8 hematopoieticprogenitor cells, derived from E12.5 fetal livers, injected into P4mouse brains, were capable of giving rise to mature parenchymalmicroglia expressing the microglia-specific marker, Tmem119.Interestingly, two separate embryonic sources of microglia havealso been recently shown to exist in zebrafish (Xu et al., 2015).Hoxb8 gene expression is highest during yolk sac hematopoiesis.

Both the later timing of the appearance of theHoxb8 lineage-labeledprogenitors in the yolk sac, and the expression of Sca-1, support thehypothesis that the Hoxb8 microglia progenitors are generatedduring the second wave of yolk sac hematopoiesis. Sheng et al.(2015) have suggested that a prominent hematopoietic populationpresent in the yolk sac during the second wave of hematopoiesis iscomposed of f-HSCs expressing Sca-1. The Hoxb8 lineage markeralso labels virtually all bone marrow-derived HSCs. BecauseHoxb8

is not expressed in adult hematopoietic stem cells (HSCs),this suggests that the Hoxb8 cell lineage marker is activated ina progenitor population that gives rise to bone marrow HSCs(i.e. f-HSCs). Our observations are consistent with the hypothesisposited by Sheng et al. (2015), that a principal hematopoieticprogenitor cell population is f-HSCs. As reported by Sheng et al.(2015) for f-HSCs, the Hoxb8-labeled hematopoietic progenitorsalso appear in the P-Sp/AGM region and fetal liver, where they aresignificantly expanded in number. That Hoxb8 expression isundetectable in the brain during embryonic development or inadult microglia further supports the hypothesis that the Hoxb8lineage in the brain infiltrates from the embryonic periphery, ratherthan arising from a subpopulation of non-Hoxb8 microglia thatsubsequently express Hoxb8 in the developing brain.

The passage of Hoxb8 microglia progenitors through theP-Sp/AGM region and fetal liver, prior to infiltration into thedeveloping brain, provides added opportunities for these microgliato acquire additional genetic and epigenetic complexity and therebypotential for a greater repertoire of functions. The second wave ofmicroglia infiltration into the brain occurs prior to maturation of theBBB and uptake of these progenitor cells would likely terminateonce the BBB is closed. As with non-Hoxb8 microglia, Hoxb8microglia are therefore within a closed environment and maintainedat steady-state concentrations in the brain through a balance of self-renewal and apoptosis.

Comparison of the RNA transcriptome of Hoxb8 microgliawith non-Hoxb8 microglia shows that the two populations haveextremely similar expression profiles. Also, two molecular markersrecently characterized as specific to parenchymalmicroglia,Tmem119and Sall1 (Bennett et al., 2016; Buttgereit et al., 2016), are equally andprominentlyexpressed in bothmicroglial subpopulations.Not only arethese two populations of microglia molecularly very similar, but theyalso show similar responses to injury and synaptic pruning. Two typesof injury responses were examined: a fast response, within 30 min of afocal laser-induced injury, and a response to more extensive damageinduced bya needle poke. TheHoxb8microglia response to the needlepoke injury appeared to be more extensive than observed for thenon-Hoxb8 microglia. Both populations of microglia respondedaggressively and similarly to acute injury by immediate extensions oftheir projections. Finally, activity-dependent postnatal pruning in theretinogeniculate nucleus was indistinguishable between the twomicroglial subpopulations.

Although these populations of microglia have much in commonmolecularly and functionally, they were also distinguishable.Interestingly, the relative concentration of Hoxb8 microglia washigh in regions of the brain previously characterized by fMRI asbelonging to the ‘OCD circuit’ (Graybiel and Rauch, 2000).The distinct distribution ofHoxb8microglia in the adult brain mightbe an important contributor to the specific dysfunctional behavioraloutput observed inHoxb8mutant mice. The mechanisms responsiblefor generating the distinct distributions of non-Hoxb8 microglia andHoxb8microglia, whether passive or active, has not been determined.

Additional functional differences between the two subpopulationsof microglia could emerge. It appears that the effects of the Hoxb8mutation on Hoxb8 microglia, which comprise ∼25% of total brainmicroglia, cannot be compensated for by the presence of non-Hoxb8microglia (∼75% of total microglia) or the mouse would not exhibitan aberrant behavioral pathology associated with this mutation.

An association of a broad spectrum of neuropsychiatric disorderswith immune dysfunctions is well documented in the literature(Ashwood et al., 2006; da Rocha et al., 2008; Hounie et al., 2008;Kronfol and Remick, 2000; Lang et al., 2007; Leonard and Myint,

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2009; Strous and Shoenfeld, 2006). Further, among genes revealedby genome-wide association studies of multiple neuropsychiatricdisorders are a common class of genes known for their prominentroles in immunology (International Schizophrenia et al., 2009;Shi et al., 2009; Stefansson et al., 2009). What was unclear from theabove reports was which sets of genes are causative. In our case, wehave argued for the hypothesis that defective Hoxb8 microgliaappear causative for a specific behavioral dysfunction. Asidefrom Hoxb8 microglia being in the appropriate regions of theadult brain, why is the behavioral output inHoxb8mutant mice sospecific? In a separate study, we have shown that Hoxb8 mutantmice also exhibit high levels of anxiety, and these mutant miceresponded favorably to serotonin uptake blockers such asfluoxetine (Nagarajan et al., 2017). High levels of anxiety havealso been associated with multiple neuropsychiatric disorders,particularly obsessive-compulsive-spectrum disorders. Patientswith OCD, during periods of high anxiety, often participate intheir pathological compulsive activity, whereas when they are notanxious, they can control their compulsive behavior (Markarianet al., 2010). A reasonable argument can be made that particularlyamong social animals like humans and mice, one of the multipleroles for microglia is to maintain brain homeostasis, includingprotection from excessive anxiety. Failure of such maintenancemay lead to behavioral disorders, with the particular disorder beingdependent upon the constellation of genetic and environmentalinsults experienced by the individual patient. Regardless of the targetsof microglia for maintaining homeostasis, what is clear is that we arein the midst of a glia revolution with the recognition that glia havemuch broader roles in brain function, brain homeostasis and brainpathology than we ever imagined.

MATERIALS AND METHODSAnimalsGt(ROSA)26Sortm14(CAT-tdTomato)Hze (Ai14, #007908), Cx3cr1GFP mice(#005582) and Csf1rtm1.2Jwp (#021212) mice were obtained from theJackson Laboratory. Hoxb8IRES-Cre mice were generated in our laboratoryand reported in Chen et al. (2010). Runx1IRES-GFPmicewere a kind gift fromDr James Downing, Department of Pathology, St. Jude Children’s ResearchHospital, Memphis, TN, USA.

Embryo isolationTimed matings were set up between male and female mice of the requiredgenotypes. The uterine horns were removed and placed on ice-cold 1× PBS.The embryos were isolated from the uterus with the help of sharp forceps,washed in ice-cold 1× PBS and incubated in 4% (wt/vol) paraformaldehyde(PFA; EMS 15713) in 1× PBS overnight. Embryonic tissues/brains werethen processed for immunohistochemistry as described below.

ImmunohistochemistryFor adult brain isolation, mice were overdosed with avertin andtranscardially perfused with 4% PFA. The brains were isolated andincubated in 4% PFA for 2 h on a bench-top shaker at room temperature.Embryonic and adult samples were kept in 10% sucrose overnight at 4°C ona shaker, followed by another overnight incubation at 4°C in 30% sucroseuntil brain tissues sink to the bottom. These samples were then embedded inTissue-Tek OCT (Sakura 4583), frozen on liquid nitrogen and stored at−80°C. For immunohistochemistry, brain samples were sectioned at 20-25 μm using a Leica CM1900 cryostat and mounted on positively chargedmicroscope slides (Fisherbrand Tissue Path SuperFrost Plus Gold slides, 22-035813, Fisher Scientific). Sections were washed with 1× PBS andpermeabilized with 0.2% Triton X-100, 1% sodium deoxycholate solution,then incubated overnight with primary antibody mixture at 4°C. Thefollowing day the sections were washed and incubated with secondaryantibodies for 2 h at room temperature. Finally, the sections were stained

with 4′,6-diamidino-2-phenylindole (DAPI; D1306, Molecular Probes) andmounted with ProLong Gold antifade reagent (P36934, Molecular Probes)and microscope cover glass (Fisherbrand 22-266882). Images were acquiredon Leica TCS SP5 confocal microscope and processed and analyzed usingImaris 7.7 (Bitplane), as described below.

Antibodies for immunohistochemistryPrimary antibodiesPrimary antibodies were as follows: chicken anti-GFP (1:500, GFP-1020,Aves Labs), rabbit anti-RFP (1:500, 600-401-379, Rockland), rat anti-RFP(5F8) (1:250, 5f8-100, Chromotek), guinea pig anti tdTomato (1:250, AB2631185, Frontier Institute), rabbit anti-cleaved caspase-3 (Asp175) (1:500,9661, Cell Signaling Technology), rat 4D4 antibody (1:200, provided by DrOleg Butovsky, Harvard Medical School), rabbit anti-Tmem119 (28-3)(1:500, 209064, Abcam).

Secondary antibodiesSecondary antibodies were as follows: goat anti-chicken Alexa Fluor 488(1:500, A-11039, Thermo Fisher Scientific), goat anti-rabbit Alexa Fluor 555(1:500, A-21428, Thermo Fisher Scientific), goat anti-rabbit Alexa Fluor647 (1:500, 111-605-144, Jackson ImmunoResearch), goat anti-rat AlexaFluor 555/647 (1:250, A-21434/A-21247, ThermoFisher Scientific) and goatanti-guinea pig Alexa Fluor 555 (1:250, A21435, Thermo Fisher Scientific).

Both the primary and secondary antibodies used have been monitored forspecificity and cross-reactivity.

EdU labelingTo label proliferating microglia at different time points (P0, P4, P8,P16 and P32, n=3 per time point), Cx3cr1GFP/+; Hoxb8IRES-Cre/+;ROSA26CAG-LSL-tdTomato/+ mice were anesthetized with isoflurane andinjected (dosage 10 µl/gm body weight) with 10 mM EdU (A10044,Molecular Probes), twice every 1.5 h. After the final incubation period thebrains were isolated and processed for immunohistochemistry and confocalimaging as described above. The EdU-labeled cells were identified using theClick-iT EdU Alexa Fluor 647 Imaging Kit (C10340, Molecular Probes).

Embryonic tissue processing for FACSEmbryos were removed from the uterus and placed on ice in 5% fetal bovineserum (FBS, Atlanta Biologicals) in 1× Hanks’ balanced salt solution(HBSS, Gibco). Dissections of the following tissues were performed at theirrespective time points: yolk sac (E7.5, E8.5, E9.5, E10.0, E10.5, E11.5,E12.5), P-Sp/AGM region (E9.5, E10.0, E10.5, E11.5), fetal liver (E10.0,E10.5, E11.5, E12.5, E13.5, E14.5, E15.5, E18.5) and bone marrow (P35).Dissected tissues before E12.5 were pooled together to obtain enough cellsat these time points for flow cytometry. The tissue was broken up into singlecells using a 26G needle. The dissociated tissues were spun down at 1500 gfor 5 min at 4°C. Cells were washed three times with 1× HBSS and spundown at 1500 g for 5 min. To obtain a single-cell suspension, the cells werepassed through an 80 µm mesh. The suspension was incubated in 20 µl ofred blood cell lysis buffer (0.15 M ammonium chloride, 1 mM KHCO3) for5 min, then ∼13 ml of HBSS was added and incubation continued for 5 minto lyse red blood cells, followed by another 5 min spin at 200 g. The anti-mouse antibodies used consisted of the following: TER-119 PerCP-Cy5.5(1:50, 116228, BioLegend), CD41 APC-eFluor 780 (1:100, 47-0411-82,eBioscience), CD45 APC (1:100, 103112, BioLegend), c-Kit PE-Cy7(1:100, 105814, BioLegend), Sca-1 APC (1:100, 108112, BioLegend), Sca-1 APC/FIRE 750 (1:100, 108146, BioLegend), Lin-1 Alexa Fluor 700(1:100, 133313, BioLegend) in 1× HBSS. Cells were incubated in the darkwith their respective antibody cocktail for 30 min on ice. Cells werewashed with 1× HBSS and spun down at 1500 g for 5 min, thenresuspended in 1× HBSS with DAPI (1:500). Flow cytometry data wereobtained using the BD Bioscience FACSCanto II flow cytometry sorter.All FACS data were analyzed with FlowJo 10.0.7 (Celeza).

FACS gating strategy for embryonic and adult tissue populationsFor all embryonic tissue populations, live cells were gated with side scatter(SSC) and forward scatter (FSC) parameters. Viability and singlets were

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gated with FSC and DAPI parameters. For Cx3cr1GFP/+; Hoxb8IRES-Cre/+;ROSA26CAG-LSL-tdTomato/+ triple-transgenic mice, the following gates wereapplied depending on the developmental stage of the embryo: time pointsfor yolk sac and P-Sp/AGM region (E7.5, E8.5, 9.5, E10.0, E10.5, E11.5)included CD41 and TER119 (Ly76) parameters in order to excludeplatelets, red blood cells and erythrocytes, and select CD41+ or CD45+

hematopoietic cells. These cells were further analyzed for tdTomato andc-Kit or Sca-1 to examine the percentage of tdTomato+ cells in thehematopoietic progenitor or f-HSC compartment. Time points for fetal liver(E10.0, E10.5, E11.5, E12.5, E13.5, E14.5, E15.5, E18.5) were gated in asimilar fashion except that CD45was used to gate CD45+ hematopoietic cells.For bone marrow, viable cells (DAPI−) were gated for Lin-1−, c-Kithigh andSca-1+ to identify the HSC cell population to examine the percentage oftdTomato+ cells in the HSC compartment. For Runx1IRES-GFP+/−;Hoxb8IRES-Cre+/−; ROSA26CAG-LSL-tdTomato/+ compound transgenicmice: TER119− CD41+ cells were selected and gated with c-Kit toidentify hematopoietic progenitors. Hematopoietic progenitors were furtheranalyzed to examine the percentage of tdTomato+ cells that were GFP+.

Postnatal brain injectionsHematopoietic progenitor cells were isolated (described above) from E12.5fetal livers of Cx3cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+

embryos and sorted by flow cytometry using the BD Bioscience FACSARIA flow cytometry sorter. Sorted Hoxb8 lineage progenitor cellsconsisted of the following immunophenotype: DAPI− TER119− c-Kithigh

tdTomato+. Freshly sorted cells were directly injected bilaterally into themotorcortex (12.5-25K cells/hemisphere) of wild-type (C57Bl6/J) or Csf1r−/−

newborns (P0-P4). These brains were harvested, sectioned and stained withantibodies against tdTomato,GFPandTmem119, fromP14 toP17 to examinewhether fetal liver-derived Hoxb8 lineage hematopoietic progenitor cellsinjected into P0-P4 pups can colonize the postnatal brain and becomeHoxb8 microglia. Microglia were observed in seven of nine brainsinjected.

qRT-PCRTotal RNA was isolated from either yolk sac (E8.5, E9.5, E10.5, E11.5,E12.5), P-Sp/AGM region (E10.0, E11.5), fetal liver (E11.5, E12.5,E13.5) or head/brain (E9.5, E10.5, E11.5, E12.5, E13.5, E18.5), andpurified using the RNeasy Kit (Qiagen). The concentration and quality oftotal RNAwas determined by using a NanoDrop2000c spectrophotometer(Thermo Fisher Scientific), and 500 µg of total RNAwas used for reversetranscription into cDNA using the SuperScript III First-Strand SynthesisSuperMix (Invitrogen). For qRT-PCR, an Applied Biosystems 7900HTinstrument was used to amplify 50 ng Hoxb8-specific cDNA and 50 ngGapdh-specific cDNA (an endogenous housekeeping control) using aHoxb8 probe (Thermo Fisher Scientific) and a Gapdh probe (ThermoFisher Scientific), respectively. Each tissue sample was run in triplicate.Hoxb8 gene expression was quantified using the comparative CT method(ΔCT) by normalizing to Gapdh expression in age-matched tissues.

Microglia isolationCortical microglia were isolated from four independent ∼12-week-old two-color animals (Cx3Cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+).Animals were humanely euthanized by isoflurane, cortical tissue dissectedand meninges removed, minced and trypsin digested (1% trypsin, 37°C,30 min in HBSS). Cortical single-cell suspensions were generated bymechanical trituration and successive filtration (70 and 30 µm mesh) andglia enriched via capture with anti-Cd11b (Itgam) magnetic beads (MiltenyiBiotec). Bead-associated cortical microglia were then sorted on a BDFACSAriaII gating on tdTomato and GFP with collection into RNAlatersolution, flash frozen and stored at −80°C.

RNA sequencing isolation and analysesTotal RNA was isolated and cloned by the University of UtahHigh-throughput Genomics Core using a Clontech UltraLow RNAisolation kit, and barcoded libraries sequenced on an Illumina HiSeqplatform (50 bp nonstranded single-end reads), resulting in ∼25-30

million sequences per biological replicate. Sequence quality wasconfirmed by FastQC. Raw fastq reads were mapped using STAR(2.4.1c) (Dobin et al., 2013). Indices for STAR were built based upon theGencode GRCm38v3 release M5 of the mouse genome. The index forSTAR was constructed based upon the primary assembly and includedsplice junction information from the GTF file. The cDNA sequencescorresponding to Cre, tdTomato and eGFP were appended to the fasta filesprior to index generation. Mapping was accomplished using defaultparameters for STAR necessary to the utilize splice junction informationbuilt into the corresponding index. STAR read counts at the gene levelwere generated using feature counts from the SubRead package.

Deseq2 analyses were conducted independently on all count data sets tocompare relative transcript levels between the green (non-Hoxb8, control)and yellow (Hoxb8, experimental) microglia data sets. Deseq2was run usingdefault parameters and differentially expressed genes determined based upona padj cutoff value <0.1. The list of differentially expressed genes wasgenerated (Table S1).

Microglial activation using focused laser ablationLive brain imaging was performed using a Prairie Technologies UltimaMultiphoton Microscopy System. For live imaging in the cerebral cortex,an open skull window was prepared on 1- to 2-month-old Cx3cr1GFP/+;Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+ mice. The open skull windowprep was performed according to the previously described protocol byPozner et al. (2015). The mice were mounted on a custom-built head frameand imaged under 1.5% isofluorane anesthesia. Imaging was performedwith a Chameleon Ti:Sapphire laser at 950 nm and a 16× water-immersionobjective (0.8 NA, Nikon) with 3× optical zoom. The signals were acquiredusing GaSP detectors with 490-560 nm bandpass filter for GFP and 570-620 nm bandpass filter for tdTomato. Microglial cells were imaged 50-80 μmbelow the pial surface at 1024×1024 pixel resolution and a sampling rate of0.05 frames/s for 30-35 min. Focused laser injury was induced at a small area(8×8 pixels) within the region of interest that was scanned at a frequency of1 Hz for 2 min with the laser power increased by 75%. Image acquisition wasachieved using the Prairie View 5.2 software and images were analyzed usingImaris (Bitplane).

Microglial activation using intracranial injectionsTo assess microglial activation response, we induced focal injury in the motorcortex of 3- to 4-month-old Cx3cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+ mice. Microglial activation was induced by a needle poke injurythrough stereotaxic intracranial injections. Mice were anesthetized withisoflurane (4% for induction, 1.5% for surgery) and their heads were mountedon a stereotaxic frame (Kopf). Anesthetized mice were placed on a heatingpad (FHC) to regulate and maintain their body temperature at 37°C, and thedepth of anesthesia was tested and monitored by toe-pinch reflex andrespiration rate. A subcutaneous injection of 50-60 µl of 0.5% marcaine(Hospira) was applied as local anesthesia at the incision site. An incision of∼7 mm was made along the anterioposterior axis to expose the skull. Theskull was cleaned with ferric chloride solution and saline, and dried withsterile cotton tipped applicators. Using a high-speed dental drill, a burr holewas created at the stereotaxic position anteroposterior (AP) −1.5 mm;mediolateral (ML) −1.9 mm; dorsoventral (DV) −1 mm, using Bregma asreference. Tissue damage was induced by intracranial injection of 0.1 μl ofsterile 1× PBS, delivered at a rate of 20 nl/min using a QuintessentialStereotaxic Injector (QSI) (Stoelting). Following the injection, saline wasapplied to the exposed skull and skin, then the incisionwas sutured. Seven daysfollowing the injection, the mice were humanely euthanized by isoflurane, andthe brains were isolated and analyzed with immunohistochemistry andconfocal imaging.

Synaptic pruning assayTo study the pruning behavior of each microglial population, P4pups (Cx3cr1GFP/+; Hoxb8IRES-Cre/+; ROSA26CAG-LSL-tdTomato/+) wereadministered bilateral intravitreal injections of 1 mg/ml Alexa Fluor 647-conjugated cholera toxin B subunit (CTB-A647, C34778, MolecularProbes). After 24 h, the mice were humanely euthanized by isoflurane, their

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brains were harvested, then fixed in 4% PFA, passed through the sucrosegradient, embedded in OCT, and stored at −80°C. Cryosections (25 µm)were collected for immunohistochemistry to fluorescently label the Hoxb8and non-Hoxb8 microglia, as described above. Brains chosen for analysiswere those that exhibited sufficient dye fills in the dorsal lateral geniculatenucleus (dLGN).

Confocal imaging parametersImages were acquired on Leica TCS SP5 confocal system. The imagingparameters used for these different types of analyses are described below.

Microglia countingFor density analysis, the brain sections were imaged with a 10× objective(0.4 NA, Leica) and 1.5× digital zoom, acquired at 512×512 resolution and400-600 Hz scan speed, using a 5.0 µm z-depth through the tissue.

Synaptic pruning analysisConfocal images of the dLGN regions of the thalamus were acquired with a63× oil immersion objective (1.40-0.60 NA, Leica), at 1024×1024resolution, 0.5 µm z-depth.

Imaris image analysisThe images acquired with the confocal and two photon microscopy wereprocessed using Imaris Image Analysis Software ×64 (v 7.7.2, Bitplane).The parameters used for each type of analysis are mentioned below.

Microglia countingTo count the number of microglia per unit area (mm2), the ‘Spots’ functionwas used. To further identify subsets of cells that co-label with anothermarker (proliferating cells, apoptotic cells, etc.), the spots were filteredusing ‘mean intensities’ of the fluorescence of the marker. To quantify thearea of the region of analysis, ‘Surface’ function was used.

Movement of microglial processesTo assess the speed of the microglial processes moving towards the site offocused laser injury (see experiment above), at first, the site of injury wasidentified with ‘Surfaces’ to set it as the point of reference. This was followedby performing the following Imaris Xtension functions: ‘Time subtractaverage’ to exclude the stationary site of injury from the subsequentmovement/tracking calculations, and ‘Drift correction’ to correct for anyartifacts caused by the movement of the test subject (owing to breathing, etc.)during image acquisition. To follow the motion of the microglial processes,the microglia were identified with ‘Spots’ (the bulbous ends of the processes).Xtension ‘Autoregressive motion’ identified the movements (tracks) of thesechosen processes through the entire time period. Following that, runningthe Xtension ‘Distance transformation’ on the site of injury surface finallyproduced the numerical values of the different parameters, such as speedand distance, that were used for the analysis of the movement of themicroglial processes.

Synaptic pruning analysisFirst, each microglial cell surface/cellular volume was isolated using‘Surface’ function, and the CTB-A647 puncta were identified using ‘Spots’function. The numbers of CTB-A647 spots that co-localize with themicroglia were quantified using the Imaris XTension ‘Split into surfaceobjects’.

Statistical analysisUnless otherwise stated, all results are reported as mean±s.e.m. andstatistical tests were deemed significant when P<0.05. Statisticalcalculations (Student’s t-test) were performed with GraphPad Prism 6.0(GraphPad Software).

AcknowledgementsWe thank our tissue culture team, S. Barnett and C. Lenz; animal husbandry supportteam, K. Lustig, J. Wangerin, R. Beglarian, R. Focht, K. Prettyman, J. Hayes,M. Rudd and K. Smith-Fry; K. Gilleese for the preparation of the manuscript; and

all members of the Capecchi laboratory. Sequencing was performed at the HighThroughput Genomics Core Facility, University of Utah. We thank M. Vetter,Department of Neurobiology and Anatomy, University of Utah, for providingCx3cr1GFP and Csf1r−/− mice; B. Barres, Stanford School of Medicine, for providingthe anti-Tmem119 primary antibody; and O. Butovsky, Center of NeurologicDiseases, Harvard Medical School, for providing the 4D4 primary antibody.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: S.D., D.V.D., A.B., S.T., M.R.C.; Methodology: S.D., D.V.D.,E.P., A.B., S.T., M.R.C.; Software: S.D., D.V.D., M.H.; Validation: S.D., D.V.D., E.P.,M.H.; Formal analysis: S.D., D.V.D., M.H.; Investigation: S.D., D.V.D., E.P.;Resources: S.D., D.V.D., E.P.; Data curation: S.D., M.H.;Writing - original draft: S.D.,D.V.D., M.H., M.R.C.; Writing - review & editing: S.D., D.V.D., A.B., S.T., M.R.C.;Visualization: S.D., M.R.C.; Supervision: A.B., S.T., M.R.C.; Project administration:S.T., M.R.C.; Funding acquisition: M.R.C.

FundingThis work was supported by the Foundation for the National Institutes of Health [R01MH093595 to M.R.C.], the National Cancer Institute [5P30CA042014-24], theNational Center for Research Resources [1S10RR026802-01] and the University ofUtah Flow Cytometry Facility. Deposited in PMC for release after 12 months.

Data availabilityRNA-seq data have been deposited in GEO under accession number GSE124710.

Supplementary informationSupplementary information available online athttp://dev.biologists.org/lookup/doi/10.1242/dev.152306.supplemental

ReferencesAshwood, P., Wills, S. and Van de Water, J. (2006). The immune response in

autism: a new frontier for autism research. J. Leukoc. Biol. 80, 1-15.Bennett, M. L., Bennett, F. C., Liddelow, S. A., Ajami, B., Zamanian, J. L.,

Fernhoff, N. B., Mulinyawe, S. B., Bohlen, C. J., Adil, A., Tucker, A. et al.(2016). New tools for studyingmicroglia in themouse and human CNS.Proc. Natl.Acad. Sci. USA 113, E1738-E1746.

Buttgereit, A., Lelios, I., Yu, X., Vrohlings, M., Krakoski, N. R., Gautier, E. L.,Nishinakamura, R., Becher, B. and Greter, M. (2016). Sall1 is atranscriptional regulator defining microglia identity and function. Nat. Immunol.17, 1397-1406.

Chen, S.-K., Tvrdik, P., Peden, E., Cho, S., Wu, S., Spangrude, G. andCapecchi,M. R. (2010). Hematopoietic origin of pathological grooming in Hoxb8 mutantmice. Cell 141, 775-785.

Dalmau, I., Vela, J. M., Gonzalez, B., Finsen, B. and Castellano, B. (2003).Dynamics of microglia in the developing rat brain. J. Comp. Neurol.458, 144-157.

da Rocha, F. F., Correa, H. and Teixeira, A. L. (2008). Obsessive-compulsivedisorder and immunology: a review. Prog. Neuropsychopharmacol. Biol. Psychiatry32, 1139-1146.

Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut,P., Chaisson, M. and Gingeras, T. R. (2013). STAR: ultrafast universal RNA-seqaligner. Bioinformatics 29, 15-21.

Erblich, B., Zhu, L., Etgen, A. M., Dobrenis, K. andPollard, J.W. (2011). Absenceof colony stimulation factor-1 receptor results in loss of microglia, disrupted braindevelopment and olfactory deficits. PLoS ONE 6, e26317.

Ferkowicz, M. J., Starr, M., Xie, X., Li, W., Johnson, S. A., Shelley, W. C.,Morrison, P. R. and Yoder, M. C. (2003). CD41 expression defines the onset ofprimitive and definitive hematopoiesis in the murine embryo. Development 130,4393-4403.

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 mappinganalysis reveals that adult microglia derive from primitive macrophages. Science330, 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-myeloidprogenitors. Nature 518, 547-551.

Graybiel, A. M. and Rauch, S. L. (2000). Toward a neurobiology of obsessive-compulsive disorder. Neuron 28, 343-347.

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-myeloidprogenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.Immunity 42, 665-678.

13

RESEARCH ARTICLE Development (2018) 145, dev152306. doi:10.1242/dev.152306

DEVELO

PM

ENT

Holstege, J. C., de Graaff, W., Hossaini, M., Cardona Cano, S., Jaarsma, D.,van den Akker, E. and Deschamps, J. (2008). Loss of Hoxb8 alters spinaldorsal laminae and sensory responses in mice. Proc. Natl. Acad. Sci. USA105, 6338-6343.

Hounie, A. G., Cappi, C., Cordeiro, Q., Sampaio, A. S., Moraes, I., Rosario, M. C.,Palacios, S. A., Goldberg, A. C., Vallada, H. P., Machado-Lima, A. et al. (2008).TNF-alpha polymorphisms are associated with obsessive-compulsive disorder.Neurosci. Lett. 442, 86-90.

Hua, J. Y. and Smith, S. J. (2004). Neural activity and the dynamics of centralnervous system development. Nat. Neurosci. 7, 327-332.

Inlay, M. A., Serwold, T., Mosley, A., Fathman, J. W., Dimov, I. K., Seita, J.and Weismann, I. L. (2014). Identification of multipotent progenitors that emergeprior to hematopoietic stem cells in embryonic development. Stem Cell Reports2, 457-472.

International Schizophrenia Consortium, Purcell, S. M., Wray, N. R., Stone,J. L., Visscher, P. M., O’Donovan, M. C., Sullivan, P. F. and Sklar, P. (2009).Common polygenic variation contributes to risk of schizophrenia and bipolardisorder. Nature 460, 748-752.

Jung, S., Aliberti, J., Graemmel, P., Sunshine, M. J., Kreutzberg, G. W., Sher, A.and Littman, D. R. (2000). Analysis of fractalkine receptor CX(3)CR1 function bytargeted deletion and green fluorescent protein reporter gene insertion. Mol. Cell.Biol. 20, 4106-4114.

Katz, L. C. and Shatz, C. J. (1996). Synaptic activity and the construction of corticalcircuits. Science 274, 1133-1138.

Kettenmann, H., Kirchhoff, F. and Verkhratsky, A. (2013). Microglia: new roles forthe synaptic stripper. Neuron 77, 10-18.

Kierdorf, K., Erny, D., Goldmann, T., Sander, V., Schulz, C., Perdiguero, E. G.,Wieghofer, P., Heinrich, A., Riemke, P., Holscher, C. et al. (2013). Microgliaemerge from erythromyeloid precursors via Pu.1- and Irf8-dependent pathways.Nat. Neurosci. 16, 273-280.

Kronfol, Z. and Remick, D. G. (2000). Cytokines and the brain: implications forclinical psychiatry. Am. J. Psychiatry 157, 683-694.

Lang, U. E., Puls, I., Muller, D. J., Strutz-Seebohm, N. and Gallinat, J.(2007). Molecular mechanisms of schizophrenia. Cell. Physiol. Biochem.20, 687-702.

Leonard, B. E. and Myint, A. (2009). The psychoneuroimmunology of depression.Hum. Psychopharmacol. 24, 165-175.

Li, Y., Du, X.-F., Liu, C.-S., Wen, Z.-L. and Du, J.-L. (2012). Reciprocal regulationbetween resting microglial dynamics and neuronal activity in vivo. Dev. Cell23, 1189-1202.

Madisen, L., Zwingman, T. A., Sunkin, S. M., Oh, S. W., Zariwala, H. A., Gu, H.,Ng, L. L., Palmiter, R. D., Hawrylycz, M. J., Jones, A. R. et al. (2010). A robustand high-throughput Cre reporting and characterization system for the wholemouse brain. Nat. Neurosci. 13, 133-140.

Markarian, Y., Larson, M. J., Aldea, M. A., Baldwin, S. A., Good, D., Berkeljon,A., Murphy, T. K., Storch, E. A. and McKay, D. (2010). Multiple pathways tofunctional impairment in obsessive-compulsive disorder. Clin. Psychol. Rev.30, 78-88.

McGrath, K. E., Frame, J. M., Fegan, K. H., Bowen, J. R., Conway, S. J.,Catherman, S. C., Kingsley, P. D., Koniski, A. D. and Palis, J. (2015). Distinctsources of hematopoietic progenitors emerge before HSCs and provide functionalblood cells in the mammalian embryo. Cell Rep. 11, 1892-1904.

Mikkola, H. K. A., Fujiwara, Y., Schlaeger, T. M., Traver, D. and Orkin, S. H.(2003). Expression of CD41 marks the initiation of definitive hematopoiesis in themouse embryo. Blood 101, 508-516.

Mildner, A., Schmidt, H., Nitsche, M., Merkler, D., Hanisch, U.-K., Mack, M.,Heikenwalder, M., Bruck, W., Priller, J. and Prinz, M. (2007). Microglia in the

adult brain arise from Ly-6ChiCCR2+ monocytes only under defined hostconditions. Nat. Neurosci. 10, 1544-1553.

Nagarajan, N., Jones, B. W., West, P. J., Marc, R. E. and Capecchi, M. R. (2017).Corticostriatal circuit defects in Hoxb8 mutant mice.Mol. Psychiatry, doi:10.1038/mp.2017.180.

Nimmerjahn, A., Kirchhoff, F. and Helmchen, F. (2005). Resting microglial cellsare highly dynamic surveillants of brain parenchyma in vivo. Science 308,1314-1318.

Paolicelli, R. C., Bolasco, G., Pagani, F., Maggi, L., Scianni, M., Panzanelli, P.,Giustetto, M., Ferreira, T. A., Guiducci, E., Dumas, L. et al. (2011). Synapticpruning by microglia is necessary for normal brain development. Science 333,1456-1458.

Pozner, A., Xu, B., Palumbos, S., Gee, J. M., Tvrdik, P. and Capecchi, M. R.(2015). Intracellular calcium dynamics in cortical microglia responding to focallaser injury in the PC::G5-tdT reporter mouse. Front. Mol. Neurosci. 8, 12.

Prinz, M. and Priller, J. (2014). Microglia and brain macrophages inthe molecular age: from origin to neuropsychiatric disease. Nat. Rev.Neurosci. 15, 300-312.

Sanes, J. R. and Lichtman, J. W. (1999). Development of the vertebrateneuromuscular junction. Annu. Rev. Neurosci. 22, 389-442.

Schafer, D. P., Lehrman, E. K., Kautzman, A. G., Koyama, R., Mardinly, A. R.,Yamasaki, R., Ransohoff, R.M., Greenberg,M.E., Barres,B.A. andStevens, B.(2012). Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron 74, 691-705.

Schafer, D. P., Lehrman, E. K. and Stevens, B. (2013). The “quad-partite”synapse: microglia-synapse interactions in the developing and mature CNS. Glia61, 24-36.

Schulz, 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. (2012).A lineage of myeloid cells independent of Myb and hematopoietic stem cells.Science 336, 86-90.

Sheng, J., Ruedl, C. and Karjalainen, K. (2015). Most tissue-residentmacrophages except microglia are derived from fetal hematopoietic stem cells.Immunity 43, 382-393.

Shi, J., Levinson, D. F., Duan, J., Sanders, A. R., Zheng, Y., Pe’er, I., Dudbridge,F., Holmans, P. A., Whittemore, A. S., Mowry, B. J. et al. (2009). Commonvariants on chromosome 6p22.1 are associated with schizophrenia. Nature 460,753-757.

Stefansson, H., Ophoff, R. A., Steinberg, S., Andreassen, O. A., Cichon, S.,Rujescu, D., Werge, T., Pietilainen, O. P., Mors, O., Mortensen, P. B. et al.(2009). Common variants conferring risk of schizophrenia. Nature 460,744-747.

Stephan, A. H., Barres, B. A. and Stevens, B. (2012). The complement system: anunexpected role in synaptic pruning during development and disease. Annu. Rev.Neurosci. 35, 369-389.

Strous, R. D. and Shoenfeld, Y. (2006). Schizophrenia, autoimmunity andimmune system dysregulation: a comprehensive model updated and revisited.J. Autoimmun. 27, 71-80.

Tremblay, M.-E., Stevens, B., Sierra, A., Wake, H., Bessis, A. and Nimmerjahn,A. (2011). The role of microglia in the healthy brain. J. Neurosci. 31, 16064-16069.

Xu, J., Zhu, L., He, S., Wu, Y., Jin, W., Yu, T., Qu, J. Y. and Wen, Z.(2015). Temporal-spatial resolution fate mapping reveals distinct originsfor embryonic and adult microglia in zebrafish. Dev. Cell 34, 632-641.

Zhan, Y., Paolicelli, R. C., Sforazzini, F., Weinhard, L., Bolasco, G.,Pagani, F., Vyssotski, A. L., Bifone, A., Gozzi, A., Ragozzino, D.et al. (2014). Deficient neuron-microglia signaling results in impairedfunctional brain connectivity and social behavior. Nat. Neurosci. 17,400-406.

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