Lyl-1 marks and regulates primitive macrophages and ...Sep 28, 2020 · Lyl-1 expression...

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Lyl-1marksandregulatesprimitivemacrophagesandmicrogliadevelopment

Shoutang Wang1, †, *, Deshan Ren1, ††, *, Anna-Lila Kaushik1, †††, Gabriel Matherat1, ††††, Yann

Lécluse2,DominikFilipp3,WilliamVainchenker1,HanaRaslova1,IsabellePlo1,IsabelleGodin1

1GustaveRoussy,INSERMU1287,Villejuif;UniversitéParis-Saclay,France.2PFIC,lUMSAMMICa(US23INSERM/UMS3655CNRS;GustaveRoussy,Villejuif,France3 Laboratory of Immunobiology, Institute of Molecular Genetics of the Czech Academy of

Sciences,Prague,CzechRepublic.

Presentaddresses:†DepartmentofPathologyandImmunology,WashingtonUniversitySchoolof

Medicine, St. Louis, MO, 63110, USA; ††Medical school of Nanjing university, Model Animal

ResearchCenter,NanjingUniversity,Nanjing210093,China; †††Plasseraud IP,33064Bordeaux,

France;††††INSERMU1016,CNRSUMR8104,InstitutCochin,UniversitédeParis,Paris,France

*Equalcontribution;

ShortTitle:Lyl-1inmacrophage/microgliaontogeny

Correspondingauthor:

IsabelleGodin(ORCID:0000-0001-8577-8388)

INSERMU1287;InstitutGustaveRoussy-PR1;114,rueEdouardVaillant;94805VILLEJUIFCedex;

France

Emailaddress:[email protected];

Phone:(33)142114143;Fax:(33)142115240

Keypoints:

1-Lyl-1expressionmarksyolksacmacrophagesandbrainmacrophage/microglia/BAM.

2-Lyl-1deficiencyimpairsprimitivemacrophagedevelopmentandleadstotheup-regulationof

genesinvolvedinembryopatterning.

3-Lyl-1-expressingprimitivemacrophageshaveanimmuno-modulatoryphenotype.

4-Lyl-1deficiencyimpairsmicrogliadevelopmentandtheexpressionofgenesinvolvedinneuro-

development.

preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for thisthis version posted September 28, 2020. ; https://doi.org/10.1101/2020.09.28.316570doi: bioRxiv preprint

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Abstract

During ontogeny, resident macrophages (MΦs) of the nervous system emerge from

haematopoieticstemcell-independentprogenitorsoriginatingintheYolkSac(YS),sothatfactors

impairing YS MΦ development may lead to neurodevelopmental disorders resulting from

defectivebrainresidentMΦ.

Here we show that Lyl-1, a bHLH transcription factor related to Scl/Tal-1, marks primitive

macrophage (MΦPrim) progenitors in the YS. Transcriptomic analysis of YS MΦ progenitors

indicatedthatMΦPrimprogenitorspresentatembryonicday(E)9areclearlydistinctfromthose

present at E10. Lyl-1 bHLH disruption led to an increased production and a defective

differentiation of MΦPrim progenitors. These differentiation defects were associated with

profound modifications of the expression of genes involved in embryonic patterning and

neurodevelopment. They also induced a reduced production of mature MΦ/microglia in the

earlybrain,aswellasatransientreductionofthemicrogliapoolatmidgestationandinthenew-

born.

We thus identify Lyl-1 as a critical regulator of MΦPrim and microglia development, which

disruptionmayimpairorganogenesis,includingneurodevelopmentprocesses.


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INTRODUCTION

Amongst the components of the transcription factor network that regulate the various

featuresofhaematopoieticcells,Tal-1,Lmo2,Runx1andGata2standoutasthemajorregulators

ofhaematopoieticprogenitordevelopmentduringontogeny(PinaandEnver,2007;Wilsonetal.,

2010).Tal-1,Lmo2andGata-2belongtoatranscriptionalcomplex,whichalsoincludesthebasic

helix-loop-helix(bHLH)transcriptionfactor lymphoblastic leukaemia-derivedsequence1(Lyl-1).

Contrary to its paralog Tal-1, which is mandatory for the specification of all haematopoietic

progenitors(Curtisetal.,2012;Porcheretal.,2017),thefunctionofLyl-1duringdevelopmental

haematopoiesisremainslargelyunknown.WethereforeanalysedthisfunctionusingLyl-1LacZ/LacZ

mutant mice (Capron et al., 2006), focusing on the initial steps of haematopoietic cells

developmentintheyolksac(YS).

Duringontogeny,haematopoieticprogenitorsaregeneratedinthreesuccessivewaves(Palis,

2016). The two first occur in the YS prior toHaematopoietic StemCell (HSC) generation. This

HSC-independent haematopoiesis comprises first the primitive haematopoieticwave,with the

transientproductionofmonopotentprogenitorswithembryonicspecificfeatures.ThesecondYS

wave, called transient-definitive, provides for a limited duration progenitors (mostly erythro-

myeloid) that seed the foetal liver (FL) and produce a haematopoietic progeny that displays

definitive/adultdifferentiationfeatures.Finally,HSC,generated intheaortaregion inthethird

and definitive haematopoietic wave, immediately migrate to the FL where they mature and

amplify to ultimately provide the population thatwillmaintain lifelong haematopoiesis in the

adult(CumanoandGodin,2007;Kieusseianetal.,2012).

DuringYShaematopoiesis,MΦarisingfromthetwowavesderiveeitherfromamonopotent

MΦ progenitor in the primitive wave (Bertrand et al., 2005b; Palis et al., 1999) or from the

progressive differentiation of EMP via the production of GM progenitors, and ultimately of


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granulocytes (G) and MΦ progenitors in the transient-definitive wave in a cMyb-dependent

pathway(McGrathetal.,2015).

Intherecentyears, fate-mappingapproachesaimedatdeterminingtheembryonicoriginof

tissueresidentMΦsindicatedthatmosttissuesharbourresidentMΦsofvariousorigins,suchas

YS,FLandadultbonemarrow(reviewedin(GinhouxandGuilliams,2016;HoeffelandGinhoux,

2018)),whichcomplicatesthecharacterisationofthefunctionsofthevariousMΦsubsets.Inthe

caseof brain residentMΦs, fate-mapping analyses established that, contrary toothers tissue,

these resident MΦs (microglia and Border Associated MΦ (BAM)) develop only from MΦ

progenitorsoriginatingfromtheYS(Ginhouxetal.,2010;GomezPerdigueroetal.,2015;Kierdorf

et al., 2013; Schulz et al., 2012; Utz et al., 2020), confirming a developmentalmodelwe had

previously put forward (Alliot et al., 1999). However, thewave (orwaves) of origin remained

unclearduetothelimitedcriteriathatdiscriminateprogenitorsfromthetwoYSwaves.

Wehereshowthat,attheearlystagesofYShaematopoiesis,Lyl-1expressiondiscriminates

primitive(MΦPrim)fromtransient-definitive(MΦT-Def)MΦprogenitors.Inthebrain,Lyl-1marked

theentiremicroglia/BAMpopulationattheonsetofbraincolonisationandappearedtoregulate

microglia/BAMdevelopment.

Altogether,thesedatapointtoLyl-1asamajorregulatorofearlyembryonicMΦprogenitors

developmentandadvocateforfurtheranalysestomorepreciselydelineateLyl-1functionduring

thedevelopmentofresidentMΦinhomeostaticandpathologicalcontexts.


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RESULTS

Lyl-1expressiondiscriminatesMΦPrimfromMΦT-DefprogenitorsintheearlyYS

SinceLyl-1 isexpressedintheYSfromtheonsetofhaematopoiesis(Girouxetal.,2007),we

firstexploreditsfunctionbycharacterizingtheprogenitorsproducedbyWT,Lyl-1WT/LacZandLyl-

1LacZ/LacZYS inclonogenicassay.Todoso,YSatembryonicday (E)8weremaintained inorgan

culture for 1 day (E8 OrgD1-YS), allowing only the development of progenitors from both

primitiveandtransient-definitivewaves(Cumanoetal.,1996;Cumanoetal.,2001).Compared

toWT, the productionofMΦ colonieswas increased in Lyl-1WT/LacZand Lyl-1LacZ/LacZOrgD1-YS.

Otherwise,theclonogenicpotentialandcolonydistributionwereunmodified(Fig.1a).

Using FACS-Gal assay (Sup. fig 1a), we noticed that the entire MΦ progenitor population

(cKit+CD45+CD11b+) expressed Lyl-1 at E9. In contrast, fromE9.5 (as in E8-OrgD1-YS), twoMΦ

progenitorsubsetsdiscriminatedbyFDG/Lyl-1expressionwerepresent(Fig.1b),suggestingthat

Lyl-1 maymarkMΦPrim progenitors from the earliest wave. After E9.5, the YS harbours both

MΦPrim and MΦT-Defprogenitors from the two YS waves and these two progenitors subsets

cannotbediscriminatedbyphenotype (Bertrandetal., 2005b).We therefore investigated the

known features that discriminate the two YS waves such as the origin from monopotent

progenitorsbeginningatE7.25forprimitiveprogenitors(Bertrandetal.,2005b;Palisetal.,1999)

or, for the transient-definitive progenitors, their progressive differentiation from EMP via the

productionofGMprogenitors,andultimatelyofgranulocytes(G)andMΦprogenitors(McGrath

etal.,2015)andthedependenceoncMybexpressionfortheirproduction(Hoeffeletal.,2015;

Schulzetal.,2012).

FACS-GalassayperformedatE8(0-5S),whenonlyMΦPrimarepresent,demonstratedthatall

earlyMΦPrimprogenitors, characterisedbyaCD11b+CD31+phenotype (Bertrandetal., 2005b),

displayedFDG/Lyl-1expression(Fig.1c).MostFDG+/Lyl-1+cells(69.27%±0.33%)fromLyl-1WT/LacZ


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E8-YSco-expressedCD11bandCD31andconsistentlyproducedMΦcolonies(72.78±9.65%;n=3)

inclonogenicassays,amounting1-4MΦprogenitorsperYS,avalueconsistentwithpreviously

publisheddata(Bertrandetal.,2005b;Palisetal.,1999).

AsMΦT-DefarisefromEMPsthatalsogenerateGMprogenitorsandgranulocytes(McGrathet

al.,2015),wecharacterizedthedifferentiationpotentialofFDG+/Lyl-1+andFDG-/Lyl-1-fractions

ofmyeloidprogenitors(Ter119-cKit+CD45+CD11b+)fromE10YSinclonogenicassay(Fig.1d).All

samples produced few non-myeloid contaminants, such as EMk and EMP in similar, non-

significantamounts.SimilartoWTE9-YSMΦPrimprogenitors,E10FDG+/Lyl-1+progenitorsubset

nearlyexclusivelyproducedMΦcolonies,confirmingtherestrictionofLyl-1expressiontoMΦPrim

progenitors.Incontrast,E10FDG-/Lyl-1-myeloidprogenitorsproducedGM,GandMΦcolonies,

confirmingtheirmultipotent/transient-definitivestatus.

TherestrictionofLyl-1expression toMΦPrimprogenitorswasalsostrengthenedbyRT-qPCR

comparisonofcMybexpression:FDG-/Lyl-1-progenitorsdisplayedcMyblevelssimilartoLineage

negativeSca1+cKit+progenitorsfromE12-FL,whereas,asE9-YSMΦPrimprogenitors,FDG+/Lyl-1+

YSprogenitorsexpressedlowcMyblevels,strengtheningtheirprimitivestatus(Fig.1e).

AdistinctseparationofE9andE10MΦprogenitorswasalsoconfirmedinRNA-seqanalysisof

MΦprogenitors(CD45+CD11b+cKit+)sortedatE9,whentheYScontainsonlyMΦPrimprogenitors,

andatE10,whenitcontainsbothMΦPrimandMΦT-Defprogenitors.PrincipalComponentAnalysis

pointedtoaseparationbetweenE9andE10samplesinbothWTandLyl-1LacZ/LacZYS(PC1)anda

separationbetweenWTandLyl-1LacZ/LacZsamples(PC2)cleareratE9inframewiththerestriction

toMΦPrimprogenitorsatthisstage(Fig.1f).

E9 and E10 WT MΦ progenitors differed by the expression of 726 genes. Amongst the

differentiallyexpressedgenes(DEGs),176wereup-regulatedatE9and550atE10.Considering

thepresenceofbothMΦPrimandMΦT-DefprogenitorsinE10-YS,theDEGsfoundatthisstagemay


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reflecteitherwaves-specificdifferencesorstage-dependentchangesrelatedtothematuration

ofMΦPrimprogenitors.

Overlapping the identified DEGs to those obtained by Mass et al. (Mass et al., 2016) to

characterise EMP and E10.25-E10.5 MΦs confirmed that E9 MΦPrim progenitors were clearly

distinctfromthesetwopopulations.Whileabout5%ofE10WTup-regulatedgenesbelongedto

the EMP andMΦ signatures, these genes represented respectively 27.5% and 35.6%of these

signatures (Fig. 1g). A similar separation was also observed in Gene Set Enrichment Analysis

(GSEA) (Sup. fig. 1b).Theseobservations suggest thatwithinE10MΦprogenitors some, likely

theMΦT-Defones,retainpartoftheEMPssignature.

FocusingonthedifferenceswithE10MΦprogenitorsinaWTcontext,E9MΦPrimprogenitors

werefoundtodifferfromE10progenitorsbytheirtranscriptionfactors(TF)repertoire.E10WT

MΦprogenitorswereenrichedingenesassociatedwitherythroiddevelopment(Gata1,Gata2,

Klf1), aswellasglobingenes,bothembryonic (Hbb-bh1,Hba-x,Hbb-y)anddefinitive (Hba-a2,

Hba-a1, Hbb-bt) (Fig. 1g). E9 and E10 MΦ progenitors both exhibited a high expression of

Spi1/PU.1 compared toGata1 (Fig. 1h).Thehigher expression level of erythroid genes andof

genes involved in granulo-monocytic (Mpo, Csf2r/GM-CSF receptors, Cebp, Jun) and

megakaryocytic development (Pf4, TPO signalling) (Fig. 1g, Sup. table 1) at E10 sustains the

notion that MΦT-Def progenitors retain the expression of genes that characterize their EMP

ancestor,aswellasthemonopotentstatusofE9progenitors(Fig.1d).

Altogether, these data validate the monopotent/primitive status of E9 MΦ progenitors.

However, due to the simultaneouspresenceofMΦPrim andMΦT-Def progenitors in E10-YS, the

differential expressions of makers that are wave-specific at this stage, such as Lyl-1 for the

primitivewaveandcMybandTlr2(Balounovaetal.,2019)forthetransient-definitiveone,were


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notsignificantdespiteanettendencytoadecreaseforLyl-1andanincreaseforcMybandTlr2

(Sup.fig.1c).

Through QIAGEN’s Ingenuity® Pathway (IPA) and GSEA analyses, it appeared that MΦPrim

progenitorsweremoreactive inEicosanoidsignallingthanE10progenitors (Fig. 1i).E9MΦPrim

progenitorswerealsoenrichedintypeIinterferon(IFN)βandtypeIIIFNγsignalling(Fig.1j)and,

asaconsequence,inMHC-IIrelatedgenes,especiallyCd74(top1IPAnetwork)(Fig.1k;Sup.fig.

1d).Flowcytometryanalysesconfirmedalow,butsignificant,enrichmentofMHC-IIexpression

inE9MΦcomparedtoE10progenitors (Sup. fig.1e).Fortheirpart,E10MΦprogenitorswere

moreactiveininflammatorysignalling(Fig.1i, l;Sup.fig.1d,f-h;Sup.table1).Theywerealso

moremetabolicallyactivethanE9MΦprogenitors (Sup. table1). Inaddition,thecomplement

cascadeandphagocytosisalsoprevailedatE10(Sup.fig.1i,j).

Lyl-1deficiencyinMΦPrimleadstopatterningdefects

DuringYSdevelopment,bothMΦPrimandMΦT-DefprogenitorsoriginatefromcKit+CD31+CD45-

progenitors (C subset),whichdifferentiate intoMΦσ via3 subsets (A1 toA3) (Bertrandetal.,

2005b) (Sup. fig. 2a). When evaluating the effect of Lyl-1 deficiency at the earliest stage of

MΦPrimdevelopment,bothFACS-Galandclonogenicassayspointedtoan increasedproduction

of MΦ progenitors in E8 Lyl-1WT/LacZ compared to WT-YS (Fig. 2a), concordant with our first

observation (Fig. 1a). The high increase of the expression level of Itga2b/CD41 in Lyl-1lacZ/lacZ

MΦPrim progenitors (Fig. 2b) may reflect the elevated commitment of mesodermal/pre-

haematopoieticcellstoaMΦfate(Sup.dataandsupfig.2b-d).

Wenoticedaclear-cutmodificationoftheTFnetwork(Fig.2c) thatcontrolsdevelopmental

haematopoiesis (Wilson et al., 2010): beside the expected reduction of Lyl-1 expression, the

expressionofLmo2,atargetofLyl-1(McCormacketal.,2013),wasalsodown-regulated,while


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Tal-1up-regulationmightreflectacompensatoryrole(Curtisetal.,2012).Theconsequenceof

these changes on early stages of YS development were apparent in GSEA analyses: both

pathways andGO terms uncovered a general up-regulation of signalling pathways involved in

earlystagesofembryopatterning(Wnt,HoxandSmad)inLyl-1LacZ/LacZMΦprogenitors,aswellas

deep changes in collagen, integrin and cadherin usage (Sup. table 2). Accordingly, various

developmentaltrajectorieswereaffected(Fig.2d),withanup-regulationinE9Lyl-1LacZ/LacZMΦ

progenitors of gene sets related to "anterior-posterior pattern specification" and "anatomical

structure formation involved in morphogenesis", notably skeletal and nervous system

development.

Another patterning modification was observed when comparing Lyl-1LacZ/LacZ to WT MΦ

progenitors at E10: at this stage, GSEA and KEGG analyses pointed to the down-regulation of

genesets involved inheartdevelopment(Sup. fig.2e; Sup. table3).Theheartharboursthree

residentMΦsubsets,twoofwhichoriginatefromtheYS.Someofthefeaturesthatdifferentiate

thetwoCCR2-YS-derivedsubsets(Epelmanetal.,2014)alsocharacterizeE9MΦPrimprogenitors

(highMHC-IIexpression(Fig.1k)andlowphagocytosisability(Sup.fig.1j).Therefore,afunction

forLyl-1inheartdevelopmentmaybeconsidered.

Thesepatterningdefects,whichappeartriggeredbydefectiveMΦPrimmayberesponsible,at

leastinpart,forthesignificantdecreasedoflittersizeandincreasedperinatallethality(Sup.fig.

2f) observed in Lyl-1LacZ/LacZ mice compared to WT, which indicates the requirement for

functionalLyl-1duringvariousdevelopmentalprocesses.

DefectiveMΦPrimdevelopmentinLyl-1mutants

We nextmonitored the distribution of A1-A2 and A3MΦ subsets (Sup. fig. 2a) at E10-YS,

whenallthreesubsetsarepresent,usingtheCx3cr1WT/GFP:Lyl-1LacZ/LacZdoubleknock-instrain.The


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analysisofLyl-1expressioninA1-A2-A3subsetsfromCx3cr1WT/GFPYSatE10indicatedthatLyl-1is

expressedthroughoutMΦdifferentiation,withaleveldecreasingfromA1toA3(Sup.fig.3a).

The subset distributionwashighly impactedbyLyl-1 deficiency, leading to an increasedA1

anda reducedA2andA3pool sizes (Fig. 3a). Thus, Lyl-1appears to regulateMΦ progenitors

differentiation towards mature MΦ. This defective differentiation could originate from the

alteredcytokinesignallinguncovered inE9mutantprogenitors throughGSEAand IPAanalyses

(Fig. 3b; Sup. fig. 3b). A limited or delayed differentiation of E9 MΦPrim progenitors was

supportedbythedecreasedexpressionofPtprc/CD45,Csfr1, Itgam/CD11bandCD33 (Fig. 3c).

ThePU.1signallingpathwaywasalsodecreasedinE9Lyl-1LacZ/LacZprogenitors(Sup.fig.3c;Sup.

table4b).

Lyl-1LacZ/LacZ MΦ progenitors were also deficient in the IFN signalling that characterize E9

MΦPrimprogenitors,notably Irf8,a factor involved inthedevelopmentofYS-MΦandmicroglia

(Hagemeyer et al., 2016; Kierdorf et al., 2013) (Fig. 3d). Compared to WT progenitors,

progenitorsfromLyl-1lacZ/lacZE9-YSup-regulatedtheLXR/RXRactivationpathway(Fig.3e),aswell

asvariousmetabolicpathways,includingsomeenrichedinE10WTprogenitors(Butanoateand

steroid) (Sup. table1),andotherwhichwerenot (Fructose/mannoseandFattyacid)(Fig. 2d).

They were also less active in inflammatory signalling pathways, particularly through NFkB, a

factorknowntointeractwithLyl-1(Ferrieretal.,1999),andinTLRsignalling(Tlr4,Tlr7,Tlr8,Tlr9)

(Fig.3d;Sup.fig.3b,d-e;Sup.table4b).

TobetteridentifythecoresignatureofLyl-1deregulationtakingintoaccountthematuration

thatoccurbetweenE9andE10,weoverlappedDEGsidentifiedinLyl-1LacZ/LacZMΦprogenitorsat

bothstages.Fromthe16DEGscommontobothstages (Fig. 3f),onlyone,Fcgr2b, showedan

oppositederegulationduringdevelopment,beingfirstdown-regulatedinthemutantatE9,then

expressedathigherlevelsthanintheWTatE10.


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Lyl-1+MΦ progenitorscontributetotheearlybrainandfoetalliver.

Thefoetalliver(Kieusseianetal.,2012)andbrain(Alliotetal.,1999;Ginhouxetal.,2010)are

bothcolonisedasearlyasE9byYS-derivedresidentMΦprogenitors.Wethereforeimplemented

a FACS-Gal assay to evaluate the contribution of Lyl-1-expressingMΦPrim progenitors to these

rudiments at E10 (Fig. 4a). While E10-YS comprised both FDG+/Lyl-1+ and FDG-/Lyl-1- MΦ

progenitor and mature (F4/80+) MΦ subsets (Fig. 1b), the brain from the same embryos

essentiallyharbouredFDG+/Lyl-1+MΦprogenitorsandmatureMΦ.Incontrast,MΦprogenitors

of both phenotypes (FDG+/Lyl-1+ and FDG-/Lyl-1-) were present in the FL, as in E10-YS. MΦ

progenitorsweremoreabundantinmutantFLthaninWT(Fig.4b),asintheYS,buttheamount

ofmatureMΦwasunmodified(Datanotshown).

We next focused on brain MΦ during the colonisation stage, which occurs until E11

(Matcovitch-Natanetal.,2016).Atthisstage,microgliaandperivascular,meningealandchoroid

plexus MΦ,collectivelyreferedtoas BAMs, are all located in the brain mesenchyme and are

therefore undistinguishable (Goldmann et al., 2016; Utz et al., 2020). FACS-Gal assay

demonstratedthatthewholeF4/80+microglia/BAMpopulationexpressesLyl-1throughoutthe

settlementperiod(Fig.4c).ThepresenceofLyl-1+F4/80+microglia/BAMattheearlieststageof

braincolonisationsuggeststhatalreadydifferentiatedMΦcouldparticipatetothecolonization

step.

A lineage relationship between Lyl-1+ MΦPrim progenitors and early microglia/BAM is

thereforestronglyconsidered,basedonthetimingoftheirappearanceandonsimilarfeatures,

such as the low level of cMyb expression (Fig. 4d), concordant with the cMyb-independent

development of microglia (Kierdorf et al., 2013; Schulz et al., 2012). Such a putative lineage

relationship is also supported by our RNA-seq data. WT E9 MΦPrim progenitors expressed


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significantly lower levels ofMrc1/CD206 than E10 MΦ progenitors, as well as a significantly

higher level of Sall3 and a slight increase of Sall1 (Fig. 4e), a transcriptomic pattern that

characterisesmicroglia(Lavinetal.,2014;Massetal.,2016;Matcovitch-Natanetal.,2016).This

patternsuggeststhatE9-YSMΦPrimprogenitorsshowapartialbiastowardamicrogliasignature.

TheseobservationspointtoE9MΦPrimprogenitorsasalikelysourceofembryonicmicrogliawith

thefirststageofmicrogliadevelopmentprogramalreadyinitiatedinYSMΦPrimprogenitorsatE9.

We next examined the distribution of A1-A2 and A3 MΦ subsets in the brain of E10

Cx3cr1WT/GFP:Lyl-1LacZ/LacZembryos toassess the impactofLyl-1 deficiencyat theonsetofbrain

colonisation.Lyl-1expression levels inthebrainMΦ subsetsweresimilartothoseobserved in

theYS(Fig.4f,Sup.fig.3a).However,Lyl-1deficiencyledtoanincreasedA1andareducedA3

poolsize(Fig.4g),indicatingthatinthebrain,asintheYS,Lyl-1regulatesthedifferentiationof

MΦprogenitorstowardsmaturemicroglia/BAM.

Lyl-1inactivationimpairsmicrogliadevelopmentattwodevelopmentstages

HavingdefinedLyl-1implicationduringmicroglia/BAMsettlementinthebrain,weturnedto

later development stages, focusing on CD45lo microglia. Cytometry and database analyses

(Matcovitch-Natanetal.,2016) confirmedthecontinuousexpressionofLyl-1 inmicrogliauntil

adulthood (Sup. fig. 4a).We thereforeexamined the impactofLyl-1 inactivationonmicroglia

poolsizeduringdevelopment.MicrogliaquantificationpointedtoE12asthefirststepimpacted

bythemutation.ThearrestedincreaseofthemicrogliapoolinLyl-1LacZ/LacZbrainatE12(Fig.5a)

resultedfromareducedproliferation(Fig.5b)ratherthananincreasedapoptosis(Sup.fig.4b).

Moreover,Lyl-1deficiencyalsoprovokedmorphologicalchangesinE12Cx3cr1WT/GFP:Lyl-1LacZ/LacZ

microglia, which displayed a reduced number and extent of ramifications compared to

Cx3cr1WT/GFPmicroglia (Fig. 5c; Sup. fig. 4c, d). Interestingly, fromE14, themicrogliapool size


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returned to levels similar toWTembryos (Fig. 5a), a recovery thatmay result from the sharp

reductionofapoptosisinLyl-1LacZ/LacZmicrogliaatE14(Sup.fig.4b).

P0-P3wasidentifiedasaseconddevelopmentstagealteredinLyl-1LacZ/LacZmicroglia.Atbirth,

thecellularityofLyl-1LacZ/LacZbrainwassignificantlydecreasedcomparedtoWT(Fig.5d),which

wasnotthecaseatearlierstages(Sup. fig.4e).TherecoveryofCD11b+cellswasalsoreduced

(WT: 140.96±0.91x103; n=9; Lyl-1LacZ/LacZ: 87.18±0.37x103; n=9). Consequently, Lyl-1 deficiency

triggeredanearly2-foldreductionofthemicrogliapopulation(Fig.5d). Thisperinatalreduction

ofthemicrogliapoolappearedtransient,sincenodifferencewithWTbrainwasobservedinthe

adult (Sup. fig. 4f). At perinatal stages, the reduction of brain cellularity in Lyl-1LacZ/LacZ mice

points toLyl-1asapossibleregulatorofthetrophicfunctionofmicrogliaonbraincells(Antony

etal.,2011;Uenoetal.,2013).

TheidentificationofE12andP0-P3askeystagesforLyl-1functioninmicrogliadevelopment

was confirmed by RT-qPCR analyses of the expression of genes essential for MΦ (Spi1/PU.1,

Csf1r,Mafb)and/ormicroglia(Runx1,Cx3cr1,Irf8)developmentandfunction,aswellasknown

regulatorsofdevelopmentalhaematopoiesis(Tal-1,Lmo2,Runx1)andrelatedfactors(Tcf3/E2A,

Tcf4/E2.2) (Fig. 5e, f; Sup. fig. 4g). Time course analyses highlighted the down-regulation of

Csf1r, Irf8 and Lmo2 in Lyl-1LacZ/LacZ microglia at both E12 and P0-P3, while Cx3cr1 was only

decreasedatE12(Fig.5g).NotethatLyl-1expressionwasunmodifiedinCx3cr1GFP/GFPmutants

(Fig. 5g). Interestingly,Cx3cr1,aswellas Irf8andLmo2,belongtopotentialLyl-1 targetgenes

(Wilsonetal.,2010).

Mafb expression levels in Lyl-1LacZ/LacZ microglia transiently decreased at P0-P3 and later

returned back toWT expression levels (Fig. 5h). AsMafb represses residentMΦ self-renewal

(Soucieetal.,2016),thistransientdecreasemaybelinkedtotherecoveryofanormalamountof

microglia after perinatal stages. Spi1/PU.1, Tcf3/E2A and Tcf4/E2.2 expression levels were


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unmodifiedinLyl-1LacZ/LacZmicrogliaatanystage,whileRunx1expressionwasonlyaffectedafter

birth.TheexpressionofTal-1wasdecreasedatE14andincreasedafterbirth,suggestingthatthis

Lyl-1 paralog (Curtis et al., 2012) does not compensate for Lyl-1 deficiency during embryonic

stagesofmicrogliadevelopment,butmaydosoduringpostnatalstages(Sup.fig.4g).

Remarkably, our RNA-seq results indicate that the expression level of some of the genes

deregulatedinLyl-1LacZ/LacZmicrogliaatE12andinthenew-bornwerealsodown-regulatedinLyl-

1LacZ/LacZMΦprogenitorsatE9(Csfr1:Sup.fig.3c,Lmo2:Fig.2c;Irf8:Fig.3d;Cx3cr1:Fig.5i).The

de-regulationof these genes inmicrogliawas transient, however in both locations and stages

theycoincidedwithadefectiveLyl-1LacZ/LacZMΦ/microgliadifferentiation.

Othergenesenrichedinmicroglia(Fcrls,Mef2c,Maf)(CrottiandRansohoff,2016)orinvolved

inthemaintenanceofmicrogliahomeostasis(P2ry12)(Bishtetal.,2018)werealsoexpressedin

E9Lyl-1LacZ/LacZMΦprogenitorsatalowerlevelthanintheWT,withexceptionofLpr8andAif-

1/Iba1whichexpression levelswere increased (Fig. 5i).Thederegulationof thesegenesmight

underlietherelationshipbetweenMΦPrimprogenitorsandmicroglia/neuraldevelopmentwhich

became apparent upon Lyl-1 inactivation considering the large number of neural signalling

pathwaysup-regulated in E9 Lyl-1LacZ/LacZMΦ progenitors (Fig. 2d) and the relationshipof the

DEGsenrichedinE9MΦPrimprogenitorswithbrainformationandneuro-developmentuncovered

inIPAanalysis(Sup.fig.4h).

Altogether, these data suggest that Lyl-1 deficiency in microglia may lead to neuro-

developmentaldefects.


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DISCUSSION

WehereidentifiedLyl-1asamarkerforYS-derivedMΦPrimprogenitorsandfoundthat,atthe

earliest stageofYSdevelopment, thisexpressiondiscriminatedMΦPrimprogenitors fromthose

producedinthetransient-definitivewaves.TheearlyexpressionofLyl-1inYSmesoderm(Giroux

etal.,2007),where itcannotsubstituteforthemandatoryfunctionof itsparalogTal-1forthe

generation of haematopoietic progenitors (Porcher et al., 2017), has already been established

(reviewed in (Curtis et al., 2012)). More recently, Lyl-1 was also identified as a regulator of

mesodermcellfateinhumaninducedpluripotentstemcells(iPSC)(Palpantetal.,2017),andits

function inthemaintenanceofprimitiveerythroidprogenitorshasbeendescribed (Chiuetal.,

2019).ParalleltoLyl-1expressionintheearliestwaveofMΦprogenitorgeneration,ourRNA-seq

data now reveal Lyl-1 as an essential regulator in the specification of YS mesoderm to a

haematopoietic fate since its invalidation affects major sets of genes involved in embryo

patterning.WhetherLyl-1roleduringtheonsetoforganogenesisisspecifictomesodermalstage

or related to a patterning function for MΦPrim progenitors remains to be determined. Lyl-1

involvementintwodiscretedevelopmentalstepsofembryogenesiswouldconstituteyetanother

similarity with its paralog Tal-1, which regulates both YS mesoderm determination to a

haematopoieticfateandthedifferentiationofprimitiveerythroidcells(Curtisetal.,2012).

Within theMΦ lineage, Lyl-1 functionduringnormaldevelopmentwould initially consist to

restrict the size of theMΦPrim progenitor pool and/or the durationof its production,which is

thought to be transient (McGrath et al., 2015), as shown by the increased size of this pool

observed in Lyl-1LacZ/LacZ E8-E9 YS. Lyl-1 would also promote MΦ differentiation, when this

processstartsatE9.5,asthemaintenanceofanincreasedsizeofMΦPrimprogenitorpoolinLyl-

1LacZ/LacZE10-YSappearstoresultfromadefectivecytokinesignalling.


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WealsodefinedasignatureforWTMΦPrimprogenitorsatE9,whichcomprisestheEicosanoid,

MHC-II and IFN signalling pathways, pointing to an immuno-modulatory function for these

MΦPrimprogenitors.Comparatively,MΦprogenitorsatE10wereratherinvolvedintheresponse

tobacteriaandviruses,throughphagocytosisandinflammatorysignalling,withthepreferential

expressionofTNF,TLRandTGFβsignallingpathways.Unfortunately,thesimultaneouspresence

ofMΦPrim andMΦT-Def progenitors at E10makes it difficult to attribute the changes of gene

expressiontoastagedependentmaturationofMΦPrimprogenitorsortoasignaturespecificto

MΦT-Defprogenitors. However it indicates thatmost pathways favoured byMΦprogenitors at

E10 were insensitive to Lyl-1 invalidation, except TLR signalling pathway that was down-

regulatedinE9mutantMΦprogenitors,comparedtoWT.

The limitationsofthecurrentLyl-1modelcall forthedevelopmentofnewmousemodelsthat

will allow lineage specific reports of Lyl-1 expression and function in endothelial or

haematopoietic lineages during discrete steps of early YS development and beyond. These

extended investigations would probably improve our knowledge of other HSC-independent

lineages,andbenefittotheEmbryonicStemCells/iPSCfieldinwhichtheproductionofdefinitive

cells types is complicated by the poor discrimination between primitive and definitive

populations.

WhenanalysingthecontributionofLyl-1+MΦprogenitorstoresidentMΦ,wedetectedtheir

presenceinFLandbrainrudimentsrightfromtheonsetoftheircolonisation.Inthedeveloping

brain, microglia/BAMmaintained Lyl-1 expression and shared with MΦPrim progenitors a low

levelofcMybexpression.Whilefate-mappinganalyseswereinstrumentaltofirmlyascribeaYS

origin to microglia (Ginhoux et al., 2010) (Kierdorf et al., 2013; Schulz et al., 2012) (Gomez

Perdigueroetal.,2015),thisstrategycouldnotascertainwhichYSprogenitorwavecontributes

to microglia (Mass, 2018; McGrath et al., 2015; Wittamer and Bertrand, 2020). Indeed, an


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independent/specific targeting of only one MΦ progenitors wave was prevented by: 1- the

similarphenotypeofMΦPrimandMΦT-Defprogenitors(Bertrandetal.,2005b);2-theshorttime-

span separating the two YS waves (E7.25 and E8.25); 3- the high variability of development

stagesof individualembryoswithinalitteratthesestage(DownsandDavies,1993)and4-the

durationofTAMinduction,nowadmittedtolastupto72h(Senserrichetal.,2018).Usinganex

vivo approaches whereby expression and potential can be examined in individually staged

embryos, we found that, at least between E8 and E10, Lyl-1 expression discriminatesMΦPrim

from EMP-derived MΦT-Def progenitors, and also marks the entire embryonic microglia/BAM

populations.

We therefore favour a model ascribing microglia origin to Lyl-1 MΦPrim progenitors, as they

preferentially expressed genes belonging to the microglia signature (Sall1 and Sall3, P2ry12,

Mef2c, Fcrls), while E10 MΦ progenitors favoured the expression of genes (Id3, Runx3),

belongingtoresidentMΦsignaturesinthelungandskin(Massetal.,2016).Eventhoughalater

contribution of MΦT-Def progenitors could be considered, provided they up-regulate Lyl-1

expressionatalaterstage,suchmodelissupportedbythelowlevelofcMybexpressedbyboth

Lyl-1+MΦPrimprogenitorsandLyl-1+microglia, inframewiththe intactmicrogliapool incMyb-

deficientmice (Kierdorfetal.,2013;Schulzetal.,2012)arguingforanoriginofmicroglia from

MΦPrim rather than MΦT-Def progenitors. Another argument comes from the zebrafish model

wherethefateofMΦPrimprogenitorsismoreeasilytracedthaninmammalsastheyarisefroma

location distinct fromother haematopoietic progenitors (Ferrero et al., 2018;Herbomel et al.,

2001). Finally, thismodel is supportedby the recent report that inmice lacking the ligand for

cKit,theimpaireddevelopmentofEMPsleadstothedepletionofresidentMΦsintheskin,lung

andliver,butdoesnotaffectthedevelopmentofmicroglia(Azzonietal.,2018).


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SomeconfusionintheidentificationoftheYSprogenitorsthatgivesrisetomicrogliastemsfrom

thefactthatbothYSwavesproducecellsfromerythro-myeloidlineages,hencethe"earlyEMP"

and"lateEMP"terminologyusedtoqualifythebrain-seedingMΦprogenitor.Themonopotent/

primitivenatureofE9MΦprogenitorswasconfirmedbyourRNA-seqdata,sothattheMΦPrim

progenitors terminology seems more appropriate than "early EMP" to qualify the primitive

progenitorsthatgiverisetomicroglia/BAM (Ginhouxetal.,2010;Utzetal.,2020),particularly

becauseMΦPrimandMΦT-Defprogenitorsclearlyconstitutedistinctentitiesthatneedtobebetter

discriminated in order to investigate their respective contribution and function in the tissues

residentMΦcontributedbytheYS.

AmongstthefeaturesthatdistinguishWTE9MΦPrimprogenitorsfromthosepresentatE10,

the enriched expression of MHC-II and the poor expression at E9 of genes related to

phagocytosisalsocharacteriseoneofthetwoYS-derivedCCR2-residentMΦsubsetsidentifiedin

theheart(Epelmanetal.,2014;Leidetal.,2016).AfunctionforLyl-1-expressingresidentMΦin

cardiacdevelopment thusappearsprobableconsideringalso thedown-regulationofgenesets

relatedtoheartdevelopmentandfunctioninE10Lyl-1LacZ/LacZMΦprogenitors.Thisobservation

reinforces the need to better characterise the respective contributions of both YS-waves to

residentMΦinthetissuesknowntoharboursYS-derivedresidentMΦ.

ByinvestigatingLyl-1functionatlaterstagesofmicrogliadevelopment,weidentifiedE12and

P0-P3askeystages impactedbyLyl-1deficiency.Thesetimepointscorrespondrespectivelyto

theendof"early-" (E9-E12)and"pre"-microglia" (E14-P9)stagesofthemicrogliadevelopment

program(Matcovitch-Natanetal.,2016).BasedonthegeneexpressionpatternofLyl-1-deficient

microgliaandthesignatureofMΦPrimprogenitorsintheearlyYS,amajorcontributionofLyl-1-

deficiency to neurodevelopmental disorders may be considered. Synaptic pruning and neural

maturation,whichcharacterisetheperinatalphaseofmicrogliadevelopment(Matcovitch-Natan


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etal.,2016),mightbeimpairedinLyl-1LacZ/LacZembryosconsideringthedefectsobservedatP0-

P3,thelaterkeydevelopmentalstageregulatedbyLyl-1.

Considering the sustainedexpressionof Lyl-1 inmicrogliaduringadulthood, and thedown-

regulationofgenesessentialformicrogliafunctioninLyl-1LacZ/LacZmicrogliaatE12andP0-P3,Lyl-

1 deregulation might contribute to various neuro-degenerative/neuro-inflammatory diseases

andpossiblyplayaroleinthedevelopmentofbraintumours.SuchaninvolvementofLyl-1inthe

developmentofneuropathiesissuggestedbyitsderegulationinvariouspathologicalmodelsof

brainmyeloidcells,uncoveredthroughtheanalysesofbrainmyeloidcellsdatasets(Friedmanet

al., 2018) (http://research-pub.gene.com/BrainMyeloidLandscape/#Mouse-gene/Mouse-

gene/17095/geneReport.html). Moreover, LYL-1 expression was also reported in human

microgliafromhealthyadultfrontalcortex(Wehrspaunetal.,2015)andsomereportshighlight

the relevance of LYL-1 deregulation in human neuro-developmental and neuro-degenerative

diseases (Colangelo et al., 2002; Thomas et al., 2006). Finally, LYL-1 belongs to the 5 genes

commonlydeletedinpatientssufferingfrom19p13.13micro-deletions,whichmightcontribute

to the neuro-developmental disabilities (Nimmakayalu et al., 2013). However, since Lyl-1 is

expressedinendothelialcells,includinginthebrain(Pirotetal.,2010),apossiblecontributionof

LYL-1-deficientendothelialcellstothesediseasesshouldbeconsidered.

Altogether, our findings reveal Lyl-1 as a key factor regulating the production and

differentiationofYSMΦprogenitors,aswellasmicroglialdevelopment.Lyl-1isonepartnersof

the haematopoietic transcription factor complex which function during developmental

haematopoiesis was the least studied. The development of new/more appropriate mouse

models should be considered in order to carefully delineate its diverse functions in other

residentMΦpopulationsandpossiblyotherhaematopoieticandcardiovascularlineages.


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MATERIALSANDMETHODS

Ethic statement.Micewere housed in the animal facility of Gustave Roussy Institute ("Plate-

formed'évaluationpréclinique", licence#H94-076-11).Allanimalexperimentswereconducted

incompliancewithFrenchandEuropeanlawsandregulations,underauthorizedproject#2016-

030-5798, approved by officially accredited local institutional animal (committee n°26) and

French"MinistèredelaRecherche"ethicsboards.

Mice and embryos. We used the following mouse strains: 1-C57BL/6 mice from Harlan or

CharlesRiversLaboratories,France,referredtoaswildtype(WT);2-Lyl-1LacZmice, inwhichan

in-frameinsertionoftheβ-GalactosidasereporterencodinggeneLacZreplacedtheHLHdomain

andtheentire3'endoftheLyl-1gene.Lyl-1miceweregenotypedasdescribedbefore(Capron

et al., 2006). Lyl-1LacZ/LacZ males were crossed withWT or Lyl-1LacZ/LacZ females to respectively

generate Lyl-1WT/LacZ or Lyl-1LacZ/LacZ embryos. This breeding scheme avoided the possible

detectionofFDG/Lyl-1expressioninmaternallyderivedMΦ (Bertrandetal.,2005b)inearlyLyl-

1WT/LacZ embryos; 3-Cx3cr1GFP mice (Jung et al., 2000). Cx3cr1GFP/GFPmales were crossed with

C57BL/6 to generate Cx3cr1WT/GFP mice/embryos or to Lyl-1LacZ/LacZ females to generate

Cx3cr1WT/GFP:Lyl-1WT/LacZmice/embryos.4-TheCx3cr1GFP/GFP:Lyl-1LacZ/LacZdoublemutantstrainwas

developed from Cx3cr1WT/GFP:Lyl-1WT/LacZ crosses. Cx3cr1WT/GFP:Lyl-1WT/LacZ and Cx3cr1WT/GFP:Lyl-

1LacZ/LacZmice/embryoswereobtainedbycrossingCx3cr1GFP/GFP:Lyl-1LacZ/LacZmales toC57BL/6or

Lyl-1LacZ/LacZ females,respectively.Toexcludematernal/placentalcellcontamination,theCx3cr1

transgenewasalwaysinheritedfromthemaleparent.

ThedayofvaginalplugobservationwasconsideredasE0.5.Pregnantfemalesweresacrificed

bycervicaldislocation.Pre-somiteembryoswerestagedaccordingtoDownsetal. (Downsand

Davies,1993).E8toE10.5embryoswerestagedbysomitecountingandthereafteraccordingto

morphologicallandmarks.


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Tissues preparation and cell counts. YS (E7.5-E10.5) and E10-FL were dissected as described

before (Bertrand et al., 2005a; Kieusseian et al., 2012). To perform cytometry analyses of the

developingbrain,twodifferentprotocolswereapplied:

1- For analyses performed at early stages (E9-E11), the whole brain were dissected and

dissociated as previously described (Alliot et al., 1999) and further cleaned from surrounding

tissues.

2- From E12 to adult stages, microglia were recovered following Percoll (P1644, Sigma)

separation, according to (Mildner et al., 2007). After Percoll purification, 100 µl of the cell

suspensionwasused for cell countingand the remaining cells for flowcytometryanalyses. To

estimate the number ofmicroglia per brain, the percentage of CD11b+CD45loF4/80+microglia

wasreportedtothecellcountrecordedforthecorrespondingsample.

Invitroculture.

Organculture:YSexplantswereplacedintoplatescontaining"CompleteOptiMEMmedium",i.e.

OptiMEMwithGlutamax (51985-042), 1%Penicillin-streptomycin,0.1%β-mercaptoethanol (all

fromThermoFisher)and10% foetal calf serum(FCS;Hyclone).YSexplantsweremaintained in

organcultureat37°C,5%CO2for1dayandarereferredtoasOrgD1-YS.

Clonogenic assay:WholeYSsuspensionorsortedcellswereplated in triplicateat respectively

3x103 or 100-150 cells/mL in Methocult® M3234 (StemCell Technologies Inc.) always

supplemented with Stem Cell factor (50ng/mL), EPO (3U/mL), IL-3 (10 ng/mL), all from

Peprotech,IL-6(10ng/mL,agiftfromSamBurstein,MaryvilleIL,USA),CSF-1(10ng/mL)andTPO

(10ng/mL,providedbyKirinBrewery,Tokyo, Japan).Culturesweremaintained inahumidified

incubatorat37°C,5%CO2andcolonieswerescoredatday5forprimitiveerythrocytesandday7

fortheotherprogenitortypes.


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Flow cytometry, FACS-Gal, proliferation andapoptosis assays.SeeSup. table 5 for the listof

antibodiesanddyesusedthroughoutthisstudy.CD31,F4/80,CD45,CD11bandcKitantibodies

wereusedtocharacterizemyeloidcells.Deadcellswereexcludedbyadding1µg/mL7-amino

actinomycin or DAPI (Sigma) before acquisition. Acquisitions were performed on a Canto II

cytometer and cell sorting using FACS-Aria III or Influx (All from BD Biosciences). Data were

analysedusingFlowJo(Treestar)software.

FACS-Galassay:Thisassay(Fieringetal.,1991;GuoandWu,2008),usedasareporterforLyl-1

expression,allowstheflowcytometrycharacterizationofcellsthatdisplayaβ-Galactivity,using

its fluorescent substrate (Fluorescein di-β-galactopyranoside (FDG) F1179; Molecular probe;

ThermoFisher).

Apoptosis assays: For apoptosis analysis,microglia were stained with anti-CD45-PECy7, anti-

CD11b-APC-eFluor®780andanti-F4/80-APC,washedand incubatedwithAnnexinV-FITC.7AAD

wasaddedbeforeacquisition.

Proliferation assay (BrdU incorporation): PregnantWTand Lyl-1LacZ/LacZ femaleswere injected

with BrdU (10µM) 12 days after plug detection and sacrificed 2 hours later. Microglia were

isolated and stained with CD45-PECy7, CD11b-APC-eFluor®780 and F4/80-PE antibodies. Cells

were fixed,permeabilisedandtreatedwithDNase (1hourat37°C)accordingtokit instruction

(BDPharmingenNo.552598)andBrdUincorporationwasrevealedusinganti-BrdU-APC.

Brainimaging.ToassessmicrogliamorphologyinE12embryos,themidbrainwasdissectedfrom

Cx3cr1WT/GFP:Lyl-1WT/WTandCx3cr1WT/GFP:Lyl-1LacZ/LacZembryosandsectionedthroughthemidline.

After fixation in 4% paraformaldehyde overnight at 4°C, whole midbrains were washed in

phosphate-buffered saline (PBS)/0.1M glycine and incubated overnight in PBS/15% sucrose at

4°C.Midbrains were washedwith PBS+0.1% Tween and incubated 90min. in blocking buffer


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(PBS+10%FCS)at roomtemperature (RT).Midbrainsweresubsequently immuno-labelledwith

F4/80-APCovernightat4°C.Afterwashing,theywereincubated3min.inPBS+DAPI(1µg/mL)at

RTandwashed.Finally,midbrainswereplacedinthecentralwellofglass-bottomculturedishes

(P35G-1.5-10-C; MatTek, USA) filled with PBS+10% FCS. After appropriate orientation of the

sample,thewellwascoveredwitha12mm∅glasscoverslip.Imagestackswerecollectedusing

a Leica SP8 confocal microscope. Images were processed using Imaris x 64 (version 7.7.2;

Bitplane) andPhotoshop8.0 (AdobeSystems, San Jose,CA) softwares. Tounsureanunbiased

choiceof thecells imaged, taking intoaccountpossiblechanges incelldistribution inducedby

Lyl-1deficiency,wealwaysacquiredcells insimilarpositionsregardingthe landmarkset inthe

midbrainflatmount,asshowninSup.fig.4c.

RT-qPCR analyses. TotalRNAwasextracted fromsortedCD11b+F4/80+CD45lowmicrogliausing

Trizol (ThermoFisher). After cDNA synthesis using a SuperScript™ VILO™ Master Mix reverse

transcriptase (ThermoFisher), quantitative PCRwas performed using SYBR Premix Ex TaqII (Tli

RNaseHPlus,TakaraBio).ReferencegeneswereActin,HprtandTubulin.Geneexpressionswere

normalized to the value obtained from E10-YS MΦ progenitors, E12 WT Lin-Sca+cKit+ (LSK)

progenitors or E12WTmicroglia, and relative gene expression levelswere determinedby the

ΔΔCtmethod.Geneexpressionwasconsideredundetectable ifCtvalueswere>35cycles.The

sequencesoftheprimersusedareprovidedinSup.table6.

RNA-sequencing.

Sample preparation: MΦ progenitors (CD45+CD11b+cKit+) were sorted from E9 (MΦPrim

progenitor) and E10 (MΦPrim + MΦT-Def progenitors) YS pools from either WT or Lyl-1LacZ/LacZ


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24

embryos. Four biological replicates were prepared for each sample. RNA was extracted as

describedabove.

Sampleprocessing:TheRNAintegrity(RNAIntegrityScore≥7.0)wascheckedontheAgilent2100

Bioanalyzer (Agilent) and quantity was determined using Qubit (Invitrogen). SureSelect

Automated Strand Specific RNA Library Preparation Kit was used according tomanufacturer's

instructions with the Bravo Platform. Briefly, 50ng of total RNA sample was used for poly-A

mRNA selection using oligo(dT) beads and subjected to thermal mRNA fragmentation. The

fragmentedmRNAsamplesweresubjectedtocDNAsynthesisandfurtherconvertedintodouble

strandedDNAusingthereagentssuppliedinthekit,andtheresultingdsDNAwasusedforlibrary

preparation.Thefinallibrarieswerebar-coded,purified,pooledtogetherinequalconcentrations

and subjected to paired-end sequencing (2 x 100) on Novaseq-6000 sequencer (Illumina) at

Gustave Roussy genomic facility. RNA-seq data have been deposited in the ArrayExpress

databaseatEMBL-EBI(www.ebi.ac.uk/arrayexpress)underaccessionnumberE-MTAB-9618.

RNA-sequencing analysis: Quality of RNA-seq reads was assessed with FastQC 0.11.7 and

MultiQC1.5(Ewelsetal.,2016).LowqualityreadsweretrimmedwithTrimmomatic0.33(Bolger

etal., 2014). Salmon0.9.0 tool (Patroetal., 2017)wasused forquantifying theexpressionof

transcriptsusinggenesetannotationfromGencodeprojectreleaseM17formouse(Frankishet

al.,2019).TheversionoftranscriptomereferencesequencesusedwasGRCm38.p6.

Statistical analysis was performed using R with the method proposed by Anders and Huber

implementedintheDESeq2Bioconductorpackage(Loveetal.,2014).Thedifferentialexpression

analysis inDESeq2 uses a generalized linearmodel (GLM)where counts aremodelled using a

negativebinomialdistribution.Countswerenormalizedfromtheestimatedsizefactorsusingthe

median ratio method and aWald test was used to test the significance of GLM coefficients.


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25

Genes have been considered differentially expressed when adjusted p-value < 0.05 and fold-

change>2.

Data were analysed through the use of Ingenuity® Pathway Analysis (QIAGEN Inc.,

https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) (Krämer et al.,

2014), Gene set enrichment analysis (GSEA; https://www.gsea-msigdb.org/gsea/index.jsp)

(Mootha et al., 2003; Subramanian et al., 2005), Morpheus

(https://software.broadinstitute.org/morpheus/) and Venny

(https://bioinfogp.cnb.csic.es/tools/venny/)softwares.

Statistical analysis. Statistical tests were performed using Prism 7 (GraphPad) Software.

Statisticalsignificanceis indicatedbytheexactp-valueand/oras*p<0.05,**p<0.01,***p<

0.001and****p<0.0001.


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Acknowledgements.

TheauthorsthankJulienBertrandforcriticalreadingofthemanuscript.Wearegratefultothe

staffofthefacilitiesatGustaveRoussy,theanimalfacility(PFEP,UMSAMMICaUMS3655/US23,

directedbyP.Gonin), the imagingfacility (PFIC,UMSAMMICaUMS3655/US23,directedbyC.

Laplace-Builhe),thegenomicfacilitydirectedbyN.Droin,thebioinformaticsfacility(G.Meurice),

directedbyM.Deloger.

This work was supported by fundings from Institut National de la Santé et de la Recherche

Médicale to W. Vainchenker, I. Plo and H. Raslova, from Centre National de la Recherche

ScientifiqueandUniversitédeParis-Saclay to I.Godin, fromgrants INCAPLBio to I.Plo, "Ligue

NationalecontreleCancer"CertifiedTeamtoH.Raslova,“AssociationpourlaRecherchesurle

Cancer”(n°4878)toI.Godin,GustaveRoussy(TADERE17)toD.Ren,GrantAgencyoftheCzech

Republic (GACR n°19–23154S) to D. Filipp and from fellowships from “Association pour la

Recherche sur le Cancer” to A.-L. Kaushik; “Société Française d'Hématologie" to S.Wang and

ChineseScolarshipCouncilfellowshipstoS.WangandD.Ren.

AuthorInformation.

Theauthorsdeclarenocompetingfinancialinterests.

CorrespondenceshouldbeaddressedtoI.G.([email protected]).


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FIGURELEGENDS

Figure1:Lyl-1expressiondiscriminatesMΦPrimfromMΦ T-DefprogenitorsintheearlyYS

a.Lyl-1deficiencyleadstoanincreasedproductionofMΦ progenitorsintheearlyYS:Left:Clonogenic

potentialofE8OrgD1-YScells:TheproductionofMΦprogenitors (CFU-M)was increased inLyl-1LacZ/LacZ

OrgD1-YS(n=3-5,eachsamplecontaining3-6YS;mean±s.e.m.;Unpaired,two-tailedt-Test).Thesizeof

theMΦcoloniesandthecellmorphologyweresimilarforthe3genotypes(datanotshown).Right:The

distributionofotherprogenitorswithamyeloidpotential (EMPandGM)was similar inWT,Lyl-1WT/LacZ

andLyl-1LacZ/LacZE8OrgD1-YS.

b.WhileallMΦ progenitors inE9-YS (leftpanel)expressedFDG/Lyl-1,E9.5andE10-YS (middlepanels)

harbouredtwoMΦprogenitorssubsetsdiscriminatedbytheirFDG/Lyl-1expression.FDG+/Lyl-1+andFDG-

/Lyl-1-matureMΦ (CD11b+F4/80+) also coexisted in E10-YS (right). Lyl-1 expression inMΦ progenitors

was analysed by FACS-Gal assay, using the β-Gal fluorescent substrate FDG as a reporter for Lyl-1

expression. The contour plots in WT samples indicate the level of non-specific background β-Gal

activity/FDGlabelling inWTsamples.Representativeprofilesof3 independentsamples,eachconsisting

of3-4YS(SeethegatingstrategyinSup.fig.1a).

c.MΦPrim progenitors express Lyl-1. Upper panel: Flow cytometry profiles ofWT (left) and Lyl-1WT/LacZ

(middle left) E8-YS (0-3S). CD11b+CD31- MΦs (top gate) correspond to maternal MΦ

presentatthisearlystage (Bertrand et al., 2005). All CD11b+CD31+ MΦ progenitors (lower gate)

displayedFDG/Lyl-1expression.

d. FDG/Lyl-1 positive and negative myeloid progenitors produce a distinct progeny: The type of

progenitors produced by sorted Ter119-cKit+CD45+CD11b+ myeloid progenitors was determined by

clonogenicassaysusingE9WTandLyl-1WT/LacZ YS (<18S;n=7),andE10WTYS (n=15) in3 independent

experiments. At E10,myeloid progenitors from Lyl-1WT/LacZ YSwere subdivided into FDG/Lyl-1 negative

(n=15) and positive (n=12) fractions (5 independent experiments). Samples were biological replicates

comprising6-8YS.100to150cKit+CD45+CD11b+cellsperconditionwereplattedintriplicate.FDG+/Lyl-1+

progenitors essentially producedMΦ colonies, while FDG-/Lyl-1- progenitors produced also GM and G

colonies,thusbelongingtothetransientdefinitivewave.

e.RT-qPCRquantificationofcMybexpressionlevelsincKit+CD45+CD11b+MΦprogenitorssortedfromWT

E9-YS,WT and Lyl-1WT/LacZ E10-YS, aswell as from the FDG/Lyl-1 positive andnegative fractions ofMΦ

progenitorsfromLyl-1WT/LacZE10-YS.Lin-Sca+cKit+(LSK)progenitorsfromWTE12FLwereusedaspositive

control. FDG+/Lyl-1+ MΦ progenitors from E10-YS expressed cMybLow/Neg levels similar to E9-YS, which

characterize the primitive YSwave. The FDG-/Lyl-1- fraction expressed significantly higher cMyb levels,

similartoLSKcells fromE12-FL.cMybexpression levels,shownonaLog2scale,werenormalizedtothe

meanexpressionvalueobtainedforWTE10-YS,consideredas1(Unpaired,two-tailedt-Test).


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f.DifferentiallyexpressedgenesinMΦprogenitors(CD45+CD11b+cKit+)sortedfromWTandLyl-1LacZ/LacZYS

atE9andE10.Upperpanel:Unsupervisedprincipalcomponentanalysis(PCA)plotpositionedE9andE10

MΦprogenitors in two distinct groups, followed by segregation ofWT and Lyl-1LacZ/LacZ samples. Lower

panel: Volcano plot of E9 WT vs E10 WT MΦ progenitors. Red and green dots indicate genes with

statistically significant changes in expression level. (p-value <0.05, absolute fold change≥2.0) (NDE: not

deregulatedgenes;DE-Up:up-regulatedgenes;DE-Down:down-regulatedgenes).

g.Upperpanel:VenndiagramcomparingDEGsinE9WTversusE10WTMΦprogenitorstotheEMP(Left)

orMΦ signatures defined by (Mass et al., 2016) (GEO accession numberGSE81774). The number and

percentageofDEGscommontotheEMPorMΦsignaturesisshown.

Lower panel: Expression profiles of the overlapping genes identified by the Venn diagram (Heatmap

displaystransformedlog2-expressionvalues;Unpairedt-Test,two-tailed).Notethehigherexpressionat

E10ofgenesinvolvedinerythroid(Haemoglobins:Pinkarrow;Transcriptionfactors:greenarrow),aswell

asmegakaryocyticandgranulocytic-relatedgenes(bluearrow).

h.RelativeexpressionlevelsofGata1andSpi1/PU.1,asindicatedbytheirrelativeTranscriptspermillion

kilo-bases(TPM).

i. Enriched Pathways in E9 and E10 WT MF progenitors with absolute z-score ≥2, from QIAGEN’s

Ingenuity®PathwayAnalysis(IPA).Bars:minuslogofthep-valueofeachcanonicalpathway;Orangeline:

thresholdp-valueof0.05.Ratio:genesdetected/genesperpathway.

j. Expression profiles of DEGs related to IFNγ and IFNβ response, identified by g:Profiler. (Heatmap

displaystransformedlog2-expressionvalues;unpairedt-Test,two-tailed).

k.ExpressionprofilesofDEGsrelatedtoMHC-IIcomplex(Heatmapdisplaystransformedlog2-expression

values;unpairedt-Test,two-tailed).

l. Expression profiles of DEGs related to cytokine signalling (Heatmap displays transformed log2-

expressionvalues;unpairedt-Test,two-tailed).

Figure2:

a.Lyl-1regulatestheproductionofE8MΦPrimprogenitors.

Leftpanel:FlowcytometryquantificationofFDG+/Lyl-1+CD11b+CD31+MΦprogenitorsfromWTandLyl-

1WT/LacZE8-YS(0-3S).TheMΦprogenitorsubsetwasenlargedinLyl-1WT/LacZYS(right;n=3).

Rightpanel:Inclonogenicassays,lessthanoneEMPand/orGMprogenitorperE8-YSwasdetectedinWT

and mutant samples, confirming that the assay was performed at a time when EMP-derived-

MΦprogenitorswere absent. Themajorityof the25-30 coloniesper YSwere EryP (60 to 80% in the3

genotypes).ThenumberofMΦcoloniesobtainedfromE8-YS(0-3S)wasincreasedinLyl-1WT/LacZandLyl-

1LacZ/LacZcomparedtoWT(left).OtherprogenitorswereoccasionallyandrandomlyfoundintheLyl-1WT/LacZ


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FDG+/Lyl-1+ fraction including EMPs (0.81%±0.66; n=3). (n=3-5, each sample contained 5-10 YS; plots

showmean±s.e.m.;Unpaired,two-tailedt-Test).

b. The relativeexpression levels (readcounts)of theCD41codinggene Itg2b is increased inLyl-1lacZ/lacZ

MΦprogenitorscomparedtoWTatE9(unpairedt-Test,two-tailed).

c.Relativeexpressionlevelsoftranscriptionsfactorsregulatinghaematopoieticprogenitoremergencein

Lyl-1LacZ/LacZMΦprogenitorscomparedtoWTatE9(p-valuesweredeterminedbyunpaired,two-tailedt-

Test).

d.GSEApathways(Top;FDRq-Value<0.29)andGOterms(Bottom;FDRq-value<0.01)enrichedinE9Lyl-

1lacZ/lacZcomparedtoE9WTMΦprogenitors.Highlightedarethepathwaysspecificallyrelatedtoembryo

patterning(blue)andtothedevelopmentofskeletal (green)andnervoussystems(yellow).Pinkarrows

pointtochangesrelatedtometabolicpathways.

Figure3:ThedifferentiationMΦ progenitorsisdefectiveinLyl-1LacZ/LacZYS.

a.DistributionofA1-A2andA3MΦsubsetsinE10-YSfromCx3cr1WT/GFP:Lyl-1WT/WT,Cx3cr1WT/GFP:Lyl-1WT/LacZ

andCx3cr1WT/GFP:Lyl-1LacZ/LacZembryos.WhilethesizeofthewholeMΦpopulation issimilar inthethree

genotypes (Toppanel),Lyl-1deficiency leads toamodifieddistributionof theMΦ subsets (middleand

lowerpanel)withanincreasedsizeoftheA1subsetandareducedA3pool(5-12independentanalyses,

eachsamplecumulating6-8YS.Plotsshowmean±s.e.m.;Unpaired,two-tailedt-Test).

b.GSEApathwayindicatesadeficitinJak1-StatsignallinginLyl-1LacZ/LacZMΦprogenitorscomparedtoWT

atE9(NES:normalisedenrichmentscore;FDR:falsediscoveryrate).

c. Relative expression levels (read counts) of haematopoietic markers in WT and Lyl-1LacZ/LacZ MΦ

progenitorsatE9(unpairedt-Test,two-tailed).

d. Top 1 GSEA pathway indicates that the IFN signalling pathway (left) which characterize E9MΦPrim

progenitors, and particularly Irf8 (right), is defective in Lyl-1LacZ/LacZ MΦ progenitors (NES: normalised

enrichmentscore;FDR:falsediscoveryrate).

e.Fromthe53canonicalpathwaysidentifiedbyIPAintheDEGs,9wereenrichedwithanabsoluteZscore

≥ 1. Bars:minus log of thep-value of each canonical pathway;Orange line: threshold p-value of 0.05.

Ratio:genesdetected/genesperpathway.

f.Upperpanel:VenndiagramcomparingtheDEGsinE9Lyl-1LacZ/LacZvsE9WTtothoseinE10Lyl-1LacZ/LacZ

vs E10 WT MΦprogenitors. Lower panel: Expression profiles of the DEGs common to both stages

identified by the Venn comparison (Heatmap displays transformed log2-expression values; unpaired t-

Test,two-tailed).


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Figure4:

a.Leftpanel:AllMΦprogenitorsfromE10-Brain(Left)expressedLyl-1,contrarytothecorrespondingYS

(Fig. 1b right) which harbour both FDG+/Lyl-1+ and FDG-/Lyl-1- subsets. MΦ progenitors from E10 Lyl-

1LacZ/LacZFL(right)harbouredbothFDG+/Lyl-1+andFDG-/Lyl-1MΦprogenitorssubsets.Rightpanel:inE10

brain,matureMΦ(CD11b+F4/80+gate)wereallFDG+/Lyl-1+

ThecontourplotsinWTsamplesindicatethelevelofnon-specificbackgroundβ-Galactivity/FDGlabelling

inWT samples. Representativeprofiles of 3 independent samples, each consistingof 3-4Brainor 8-12

E10-FL.

b.Quantificationof cKit+CD45+CD11b+MΦ progenitors in E10-FL (plots showmean± s.e.m.;Unpaired,

two-tailedt-Test).

c. Lyl-1marks theentire F4/80+microglia/BAMpopulation from theonsetofbrain colonisation.The

rareCD11b+F4/80low-negcellspresentinthebrainatE9areFDG/Lyl-positive(TopPanel).Greyhistograms

indicatenon-specificbackgroundβ-Galactivity/FDGlevelsinWTsamples.

d.MΦ progenitorsfromE10brainexpressedcMyblevelssimilartoE9-YSMΦPrimprogenitors.RT-qPCR

quantificationofcMybexpressionlevelsincKit+CD45+CD11b+MΦprogenitorssortedfromWTE9-YSand

fromWTandLyl-1WT/LacZbrainatE10.Lin-Sca+cKit+(LSK)progenitorsfromWTE12FLwereusedaspositive

control.cMyb expression levels, shownonaLog2 scale,werenormalized to themeanexpressionvalue

obtainedforWTE10-YS,consideredas1(Unpaired,two-tailedt-Test).

e. Heatmap showing the expression profiles of theDEGs in E9WT vs E10WTMΦPrim progenitors that

mark the development of tissue resident MΦ (Heatmap displays transformed log2-expression values;

Unpaired,two-tailedt-Test).

f.RT-qPCRanalysesofLyl-1expression inA1 toA3MΦ subsets isolated fromCx3cr1WT/GFPbrainatE10.

Lyl-1 is expressed by the 3 subsets, with levels decreasingwith differentiation. Expression levels were

normalizedtothemeanvalueobtainedforCx3cr1WT/GFPYSA1progenitors(n=3).

g.DefectivedifferentiationofbrainMΦ progenitorinLyl-1mutantembryos.DistributionofA1-A2and

A3 MΦ subsets in E10 brain from Cx3cr1WT/GFP:Lyl-1WT/WT, Cx3cr1WT/GFP:Lyl-1WT/LacZ and Cx3cr1WT/GFP:Lyl-

1LacZ/LacZembryos.Thesizeof thewholeMΦpopulationwassimilar in thethreegenotypes (Toppanel),

but Lyl-1 deficiency modified the distribution of the MΦ subsets (middle and lower panel) with an

increased size of the A1 subset and a reduced A3 pool (5-12 independent analyses, each sample

cumulatingbrainsfrom6-8embryos.Plotsshowmean±s.e.m.;Unpaired,two-tailedtTest).


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36

Figure5:Lyl-1deficiencyleadstotransientreductionsofthemicrogliapoolatE12andP0-P3.

a. Quantification of themicroglia population in E12 and E14 brain showing the decreased size of the

microgliapoolatE12anditsrecoverytoanormalpoolsizeatE14.Plotsshowmean±s.e.m.;Twotailed,

unpairedt-test.

b.AreducedmicrogliaproliferationmayaccountforthereducedmicroglianumberatE12,asshownby

thetwofoldsdecrease(right)ofBrdU-labelledcellsinLyl-1LacZ/LacZ(middle)comparedtoWT(left)brains.

Plotsshowmean±s.e.m.;Twotailed,unpairedt-test.

c. At E12, Cx3cr1WT/GFP:Lyl-1LacZ/LacZ microglia displayed a reduced number and extent of ramifications

compared to theirCx3cr1WT/GFP counterpart.Bottom:Microgliamorphologywasclassified into subtypes

dependingonthenumberofmainramifications(A:none,B:2,C:3andD:>3).Top:Microgliadeprivedof

ramificationspredominated inLyl-1-deficientmicroglia.65and61cellswererespectivelyacquiredfrom

themidbrainofE12Cx3cr1WT/GFPandCx3cr1WT/GFP:Lyl-1LacZ/LacZembryos(foreachgenotype,brainsfrom12

embryoswereacquired in3 independentexperiments).Microgliawere identifiedbyCx3cr1-drivenGFP

expressionandF4/80-APCimmuno-staining.Bar=10mm.Plotsshowmean±s.e.m.;Twotailed,unpairedt-

test.

d. InLyl-1LacZ/LacZnew-borns, thecellularityof thebrainwasconsistently lower than inWT(left),andso

wastheestimatedmicroglianumber(right).Plotsshowmean±s.e.m.;Twotailed,unpairedt-test.

e.Kinetic evolutionof Lyl-1 expression levels inWTmicroglia fromembryonic stages to adulthood.An

increasedexpressionofLyl-1 fromembryonicstagestoadulthoodwasalso inferredfromtimelineRNA-

seq.data(Matcovitch-Natanetal.,2016)(GEOaccessionnumberGSE79812).

f.QuantitativeRT-PCRanalysesalsopoint toE12andP0askeydevelopmentstagesregulatedbyLyl-1.

CD11b+F4/80+CD45low microglia were isolated at sequential development stages. Bar graphs show the

kinetic of expression of genesmodified in Lyl-1LacZ/LacZmicroglia (arrowheads), normalized to themean

expressionvalueinWTE12microglia(n=3).Errorbarsindicates.e.m.Twotailed,unpairedt-test.

g.Cx3Cr1andLyl-1expressioninmutantmicroglia.TheexpressionlevelofCx3CR1,analysedasinf,was

decreased in Lyl-1 mutant at E12 (left), while Lyl-1 expression level was unmodified in CX3CR1GFP/GFP

microgliaatE12andinnew-borns(right).

h.Mafb expression in mutant microglia.Mafb expression level, analysed as in f, was reduced in the

microgliaofLyl-1LacZ/LacZnew-borns.

i.Theexpressionofgenesenrichedinmicrogliaand/oressentialfortheirfunctionarederegulatedinLyl-

1LacZ/LacZ MΦ progenitors at E9. Relative expression levels (read counts) in WT and Lyl-1lacZ/lacZ MΦ

progenitorsfromE9YS(Pvaluesweredeterminedbyunpaired,two-tailedt-Test).


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Matcovitch-Natan,O.,Winter,D.R.,Giladi,A.,VargasAguilar,S.,Spinrad,A.,Sarrazin,S.,Ben-Yehuda,

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