Regulation of monocyte cell fate by blood vessels · PDF fileRegulation of monocyte cell fate...

ARTICLE

Received 14 Mar 2016 | Accepted 8 Jul 2016 | Published 31 Aug 2016

Regulation of monocyte cell fate by blood vesselsmediated by Notch signallingJaba Gamrekelashvili1, Roberto Giagnorio1,2, Jasmin Jussofie3,w, Oliver Soehnlein4,5,6, Johan Duchene4,

Carlos G. Briseno7, Saravana K. Ramasamy8, Kashyap Krishnasamy1, Anne Limbourg9, Tamar Kapanadze1,2,

Chieko Ishifune10, Rabea Hinkel4,11, Freddy Radtke12, Lothar J. Strobl13, Ursula Zimber-Strobl13, L. Christian

Napp3, Johann Bauersachs3, Hermann Haller1, Koji Yasutomo10, Christian Kupatt4,11, Kenneth M. Murphy7,14,

Ralf H. Adams8, Christian Weber4,15 & Florian P. Limbourg1,2

A population of monocytes, known as Ly6Clo monocytes, patrol blood vessels by crawling

along the vascular endothelium. Here we show that endothelial cells control their origin

through Notch signalling. Using combinations of conditional genetic deletion strategies

and cell-fate tracking experiments we show that Notch2 regulates conversion of Ly6Chi

monocytes into Ly6Clo monocytes in vivo and in vitro, thereby regulating monocyte cell fate

under steady-state conditions. This process is controlled by Notch ligand delta-like 1 (Dll1)

expressed by a population of endothelial cells that constitute distinct vascular niches in the

bone marrow and spleen in vivo, while culture on recombinant DLL1 induces monocyte

conversion in vitro. Thus, blood vessels regulate monocyte conversion, a form of committed

myeloid cell fate regulation.

DOI: 10.1038/ncomms12597 OPEN

1 Department of Nephrology and Hypertension, Hannover Medical School, D 30625 Hannover, Germany. 2 Integrated Research and Treatment CenterTransplantation, Hannover Medical School, D 30625 Hannover, Germany. 3 Department of Cardiology, Hannover Medical School, D 30625 Hannover,Germany. 4 Institute for Cardiovascular Prevention, Ludwig-Maximilians-University, D 80336 Munich, Germany. 5 Academic Medical Center, Department ofPathology, Amsterdam University, 1105 AZ Amsterdam, The Netherlands. 6 German Centre for Cardiovascular Research, Partner Site Munich Heart Alliance,Munich, Germany. 7 Department of Pathology and Immunology, Washington University in St Louis, School of Medicine, St Louis, Missouri 63110, USA. 8 MaxPlanck Institute for Molecular Biomedicine, D 48149 Muenster, Germany. 9 Department of Plastic, Hand and Reconstructive Surgery, Hannover MedicalSchool, D 30625 Hannover, Germany. 10 Department of Immunology and Parasitology, Graduate School of Medicine, Tokushima University, Tokushima770-8503, Japan. 11 Medizinische Klinik I, Klinikum Rechts der Isar, Technical University of Munich, D 81675 Munich, Germany. 12 Ecole PolytechniqueFederale de Lausanne, School of Life Sciences, ISREC, CH 1015 Lausanne, Switzerland. 13 Research Unit Gene Vectors, Helmholtz Zentrum Munchen, GermanResearch Center for Environment and Health (GmbH), D 81377 Munich, Germany. 14 Howard Hughes Medical Institute, Washington University inSt Louis, School of Medicine, St Louis, Missouri, USA. 15 Cardiovascular Research Institute Maastricht (CARIM), 6229 ER Maastricht, The Netherlands.w Present address: Department of Cardiology, Universitatsklinikum Dusseldorf, D 40225 Dusseldorf, Germany. Correspondence and requests for materialsshould be addressed to F.P.L. (email: [email protected]).

NATURE COMMUNICATIONS | 7:12597 | DOI: 10.1038/ncomms12597 | www.nature.com/naturecommunications 1

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Monocytes are myeloid leukocytes that circulate throughblood vessels and patrol the vascular endothelium ordifferentiate into mononuclear phagocytes. Mouse

monocytes consist of two distinct subsets that differ in behaviourand function, which can be discriminated by expression of theLy6C antigen and CX3CR1 chemokine receptor in combinationwith additional surface markers1,2. So called classical or Ly6Chi

monocytes are characterized by Ly6ChiCX3CR1lo and constitutethe more prevalent subset. They develop from the recentlyidentified common monocyte progenitor (cMoP)3, which inturn is derived from the macrophage and dendritic cellprogenitor (MDP)4. Under steady-state conditions, Ly6Chi

monocytes circulate in the blood for short periods of timeand are the definitive precursors of certain tissue-residentmononuclear phagocytes, for example in the gut, skin andspleen2. When recruited to sites of inflammation throughinteraction with subsets of activated vascular endothelial cells(ECs), Ly6Chi monocytes give rise to macrophages anddendritic cells, produce inflammatory mediators and orchestratethe inflammatory response5–7. Based on functional and geneexpression studies, these monocytes correspond to a subset of‘classical’ human monocytes.

There is a second subtype of monocytes that interactsclosely and constitutively with blood vessels. They display aLy6CloCX3CR1hi signature and hence are called Ly6Clo

monocytes. In the steady-state, Ly6Clo monocytes are long-livedand remain mostly within blood vessels, where they crawl alongthe luminal side of EC to monitor blood vessels and scavengemicroparticles8, a feature shared with the human CD16þ

monocyte subset9. After endothelial injury in the kidney,Ly6Clo monocytes orchestrate EC necrosis and clearance10.Moreover, Ly6Clo monocytes also mediate IgG-dependenteffector functions and are involved in immune complex-mediated disease11,12. Ly6Clo monocytes were also suggested tocontribute to ischemic tissue repair5.

The cellular and molecular context of Ly6Clo monocytedevelopment is far from clear. Mice deficient for the transcriptionfactor Nur77 (Nr4a1), an orphan nuclear receptor, show reducedfrequency and survival of Ly6Clo monocytes13. Recent findingsfurther suggest that in the steady state, Ly6Clo monocytes developfrom Ly6Chi monocytes2. Grafted MDP and cMoP sequentiallygive rise to Ly6Chi monocytes followed by Ly6Clo monocytes3,6.After adoptive transfer of Ly6Chi monocytes, Ly6Clo monocytesare detected in the blood and bone marrow (BM) of recipients,suggesting that Ly6Chi monocytes convert to Ly6Clo monocytesin the circulation7,14. However, the tissues and molecular eventsregulating monocyte conversion are unknown.

Notch signalling is a cell to cell contact-dependent signallingpathway regulating cell fate decisions and inflammatoryresponses in the immune system15. Activation of Notchreceptors (Notch1–4 in mammals) is controlled by membrane-bound Notch ligands of the jagged (Jag) and Delta-like (Dll) genefamilies, which show different Notch receptor binding affinitiesand tissue expression patterns, thereby controlling specific Notchsignalling outcomes16,17. The cellular interactions required forNotch signalling often occur in local tissue microenvironments,or niche, in the BM and spleen18. Vascular EC are a specializedcomponent of the niche that maintain and regulate stem cells andtheir immune cell progeny by providing instructive paracrinecues, known as angiocrine factors, in part through expression ofNotch ligands19. In the BM niche ECs trigger self-renewal andrepopulation of progenitor cells through Notch ligand Jag1activating Notch1/Notch2 receptors in stem cells20,21. Similarly,expansion and aggressiveness of B-cell lymphomas is induced byan angiocrine mechanism involving endothelial Jag1 activatingNotch2 in malignant lymphoma cells22. Specialized vascular

niches are also found in secondary lymphoid organs, such as themarginal zone (MZ) of the spleen, which constitutes an ECinterface between lymphoid follicles and the red pulp.Development of MZ B cells and Esamþ dendritic cell inthe splenic niche is dependent on Notch ligand delta-like 1(Dll1)-Notch2 signalling23–25. Monocytes, which are resident inBM and spleen, express Notch1 and Notch2, and their cell fate isinfluenced by DLL1 in vitro26.

Because of the intricate relationship of Ly6Clo monocytes withEC we reasoned that blood vessels might be involved in monocyteconversion through a Notch-dependent mechanism. We hereshow that Notch2 signalling regulates conversion of Ly6Chi

monocytes into Ly6Clo monocytes, which is controlled byNotch ligand Dll1 expressed by a population of EC present inhaematopoietic niches of the BM and spleen. Thus, blood vesselsregulate monocyte conversion, a form of committed myeloid cellfate regulation.

ResultsMonocyte populations and lineage relationships. Our aim wasto study the regulation of Ly6Clo monocytes. To discriminatemonocyte subsets and monocyte progenitor populations in micewe concurrently characterized MDP, cMoP, Ly6Chi and Ly6Clo

monocytes in BM, spleen and peripheral blood (PB) withcommon and discriminating markers of monocyte types based onknown expression profiles1–3,27. This approach was tested inCx3cr1GFP/þ reporter mice28, in which monocyte subsetsexpress distinct intensities of green fluorescent protein (GFP),but also in wild-type mice (Fig. 1a, Supplementary Fig. 1a,b andSupplementary Table 1). In addition, monocyte subpopulationswere also validated in Nr4a1-GFP reporter mice29, whichdemonstrated selective GFP expression in Ly6Clo monocytes(Supplementary Fig. 2a,b). These studies confirmed theproto-typical flow cytometry and gene expression profilesreported for Ly6Chi and Ly6Clo monocytes (Fig. 1b,c)3.

Ly6Clo monocytes are reported to derive from Ly6Chi mono-cytes under steady-state conditions, based on characteristicchanges of two markers, CX3CR1 and Ly6C, observed afteradoptive transfer of Ly6Chi monocytes7,14. To confirm and extendthese findings we performed adoptive transfer studies with Ly6Chi

monocytes that were isolated from CD45.2þ Cx3cr1GFP/þ miceand intravenously transferred into CD45.1þ -recipient mice(Supplementary Fig. 3a,b). Cell fate of donor cells, distinguishedfrom recipient by expression of GFP and congenic CD45, wasanalysed by flow cytometry 2 and 4 days after transfer in BM andspleen. When analysed by GFP and Ly6C expression, transferredLy6Chi monocytes progressively and uniformly switched to aLy6Clo monocyte phenotype displaying upregulation of GFP anddownregulation of Ly6C (Fig. 1d, Supplementary Fig. 3c). Anextended marker analysis demonstrated more complex phenotypicchanges involving the progressive acquisition of CD11c andCD43 while maintaining low expression levels of majorhistocompatibility complex (MHC)-II, consistent with conversioninto Ly6Clo monocytes. These changes occurred over a period of 4days and were observed in BM and spleen (Fig. 1d). Thus, anextended phenotypic analysis confirms conversion of Ly6Chi

monocytes into Ly6Clo monocytes.

Notch2 regulates Ly6Clo monocytes in vivo. To evaluate apotential role of Notch signalling in monocyte subset regulationwe sorted Ly6Chi and Ly6Clo monocytes from the BM andanalysed Notch-related gene-expression patterns. Compared withLy6Chi monocytes, Ly6Clo monocytes had lower expression ofNotch1 but comparable Notch2 expression in messenger RNA andprotein (Fig. 2a,e,f). Furthermore, Notch-regulated genes, Hey2

ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12597

2 NATURE COMMUNICATIONS | 7:12597 | DOI: 10.1038/ncomms12597 | www.nature.com/naturecommunications


and Hes1, were markedly induced in Ly6Clo monocytes,indicating recent or on-going activation of Notch signalling inthis subset (Fig. 2a)3,30. We next wanted to confirm these findingson corresponding human monocyte subsets. Analysis of thehuman CD16þ monocytes, which are considered equivalents of

mouse Ly6Clo monocytes, revealed higher expression of HES1compared with the classical CD14þ monocytes (Fig. 2b).

We next asked whether Notch deficiency influences monocytesubpopulations. To generate mice with conditional deletions ofNotch receptors in monocytes we crossed mice bearing floxed

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12597 ARTICLE



alleles of Notch1, Notch2 or Notch1/Notch2 (refs 17,31) with amyeloid specific Cre-recombinase strain, LysMCre (ref. 32).Strains were also back-crossed onto the Cx3cr1GFP/þ reporterstrain (Supplementary Table 2). This targeting strategy wascharacterized in detail. LysM reporter analysis in LysM-eGFPmice confirmed low LysM promoter activity in progenitorpopulations, but high promoter activity in Ly6Chi and Ly6Clo

monocytes (Supplementary Fig. 4a)33. In addition, crossing theLysMCre strain to a Cre-dependent YFP reporter strain revealedselective mature myeloid targeting, which was partial in Ly6Chi

monocytes, and more efficient for Ly6Clo monocytes andgranulocytes (Fig. 2c, Supplementary Fig. 4b), confirmingprevious reports34.

Mice with conditional deletion of Notch2 (GFPþN2DMy) hadsignificantly reduced absolute and relative numbers of Ly6Clo

monocytes in BM, PB and spleen (Fig. 2d, SupplementaryFig. 5a). The defect was only observed in Ly6Clo monocytes, sincenumbers of Ly6Chi monocytes, monocyte progenitor populationsand neutrophils were unaffected, and occurred independent ofthe Gfp reporter allele or the Cre deleter allele (Fig. 2d andSupplementary Fig. 5b–d). In contrast, mice with conditionaldeletion of Notch1 showed no alteration in monocytesubsets (Supplementary Fig. 5e), while combined deletion ofNotch1/Notch2 phenocopied the single Notch2 mutants(Supplementary Fig. 5f). Altogether, these results demonstratethat monocyte Notch2 controls Ly6Clo monocyte numbers,suggesting a role in monocyte cell fate regulation.

To further investigate the selective reduction of Ly6Clo

monocytes we next characterized Notch2 receptor expression byflow cytometry in control mice and conditional mutants. Notch2was robustly expressed in MDP, cMoP, Ly6Chi and Ly6Clo

monocytes in control mice (Fig. 2e,f). In GFPþN2DMy mice,Notch2 expression was not affected in MDPs and cMoP, butsubstantially reduced in Ly6Chi monocytes, consistent with partialCre expression and activity in this population (Fig. 2e,f,Supplementary Fig. 4c and (ref. 34)). However, although Cre-mediated targeting is more effective in Ly6Clo monocytes (Fig. 2c,Supplementary Fig. 4b)34, most remaining Ly6Clo monocytes inNotch2 mutant mice retained normal Notch2 receptor expression,due to low expression of Cre (Fig. 2e,f and Supplementary Fig. 4c).This suggests that the remaining Ly6Clo monocytes in these micedevelop from Notch2-expressing (Cre-negative) Ly6Chi monocytes(Supplementary Fig. 4c). Consistent with this idea, increasing theefficiency of Notch2 deletion in Ly6Chi monocytes, by usingNotch2f/f mice carrying two alleles of LysMCre, increased the rate ofNotch2 deletion in Ly6Chi monocytes but also lead to morestrongly reduced levels of Ly6Clo monocytes (Fig. 2g,h), whileLy6Chi monocyte levels remained normal (Fig. 2g). Thus, loss ofNotch2 at the level of Ly6Chi monocytes is compatible withgeneration of Ly6Chi monocytes, but incompatible with generationof Ly6Clo monocytes. Indeed, plotting Notch2 receptor levels onLy6C monocytes against the frequency of Ly6Clo monocytesrevealed that loss of Notch2 on Ly6Chi monocytes correlated

strongly with loss of Ly6Clo monocytes (Fig. 2h). This alsosuggested that the fate of Ly6Clo monocytes is linked to Ly6Chi

monocytes through Notch2.We next wanted to test the effects of a more selective deletion

of Notch 2 within the Ly6Clo population. To this end we used aconditional CD11c-Cre transgenic approach to delete Notch2 atearly stages of Ly6Clo monocyte development, since CD11c isselectively upregulated in Ly6Clo monocytes at early stages ofconversion (Fig. 1d, Fig. 5a). Loss of Notch2 partially reduced thenumber of Ly6Clo monocytes without affecting Ly6Chi monocytes(Fig. 2i, Supplementary Fig. 4d). Altogether, this demonstrates arequirement for Notch2 in the generation and maintenance ofLy6Clo monocytes.

Physiologic consequences of monocyte Notch2 deficiency. Tostudy the consequences of Notch2 loss of function formonocyte patrolling behaviour in more detail we performedintravital microscopy of the cremaster muscle8,35. Understeady-state conditions, the number of rolling and adherentLy6Clo monocytes was greatly reduced in GFPþN2DMy mice,which confirmed the results obtained by flow cytometry(Fig. 3a,b). Furthermore, while TNF-a treatment increased therolling and adherence of Ly6Clo monocytes in control mice, thisresponse was blunted in GFPþN2DMy mice (Fig. 3a,b),demonstrating a profound impairment of the Ly6Clo monocytesubpopulation with Notch2 loss of function. This result also ruledout the possibility that mutant Ly6Clo monocytes preferentiallylocalize to blood vessel walls. In addition, the reduction of Ly6Clo

monocytes in Notch2 mutant mice was accompanied byaccumulation of an atypical cell population expressing highlevels of MHC-II and CCR2 but low levels of CD11c, CD43 andCD11a (Fig. 3c–e), a phenotype not resembling a previouslydescribed MHC-IIþ monocyte population36.

Notch2 regulates Ly6Chi monocyte conversion in vivo. Ly6Clo

monocyte deficiency could be due to increased cell death, as wasshown in mice deficient for the transcription factor Nr4a1, whichcontrols Ly6Clo monocyte numbers in part by regulatingmonocyte apoptosis13. We, therefore, tested the hypothesis thatNotch2 regulates monocyte survival. However, neither thefraction of apoptotic cells, nor the fraction of dead cells wasaltered in each of the monocytes subsets in BM, PB or spleen inconditional Notch2 mutants (Fig. 4a,b).

To address the question whether conversion of Ly6Chi

monocytes depends on Notch2 we isolated Ly6Chi monocytesfrom Cx3cr1GFP/þ control mice or conditional Notch2 mutantsand analysed the cell fate after adoptive transfer into CD45.1þ

congenic wild-type recipients. Four days after transfer themajority of recovered donor cells from control donors hadconverted into Ly6Clo monocytes, while few remained Ly6Chi

monocytes. After transfer of cells from conditional mutants,however, the fraction of donor cells that had converted into

Figure 1 | Identification of monocyte subsets and lineage relationships. (a) Monocyte subpopulation analysis strategy in PB of Cx3cr1GFP/þ mice. Initially

cells were identified based on FSC and SSC characteristics. After exclusion of doublets (on the basis of SSC-W, SSC-A) Lin�CD11bþ cells were gated from

live (7AAD�) CD45þ gate and CD117þ and GFP� populations were excluded. Remaining cells were divided into Ly6ChiF4/80lo/� (R2) and

Ly6Clo/�F4/80lo (R3) subsets. Ly6Chi monocytes were defined from R2 as CD11c�MHC-IIlo/� (red) and Ly6Clo monocytes from R3—as CD11clo

MHC-IIlo/� (blue) cells. (b) Ly6Clo monocytes are smaller, contain fewer granules than Ly6Chi monocytes and show CD11cloGFPhiCD43þ phenotype.

Numbers are mean±s.e.m. (c) Quantitative reverse transcription–PCR analysis performed in sorted monocyte subsets from BM of Cx3cr1GFP/þ mice.

Change relative to expression in Ly6Chi cells is shown (n¼ 3/6). Error bars represent s.e.m. *Po0.05, **Po0.01, ***Po0.001; Student’s t-test.

(d) Dynamics of Ly6Clo monocyte development. BM CD45.2þCD11bþGFPþ Ly6Chi monocytes were transferred into CD45.1þ recipients and their

conversion into Ly6Clo monocytes were followed in vivo. Flow cytometry analysis of recipient spleen and BM is depicted. Transferred cells are black and for

comparison, recipient CD45.1þ (first row), CD45.1þCD11bþ (second row), CD45.1þCD11bþLy6Chi (third row) cells or CD45.1þCD11bþ Ly6ChiF4/80lo

monocytes (fourth and fifth rows) are shown in blue (representative of two experiments).




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Figure 2 | Conditional deletion of Notch2 impairs Ly6Clo monocyte development. (a) Quantitative reverse transcription–PCR analysis in sorted monocyte

subsets from BM of Cx3cr1GFP/þ mice; (n¼ 6, pooled from three experiments). (b) HES1 expression in human CD14þ (classical) or CD16þ (non-classical)

monocytes from two donors. (c) Quantification of YFPþ cells in myeloid cells from LysMCreRosaYFP mice as a hallmark of Cre activity. Data are pooled from two

experiments with three mice in each group. (d) Flow cytometry of myeloid cell subpopulations in mice with conditional deletion of Notch2. Absolute number of

cells per mg BM, per ml blood or per spleen is shown (top). Relative frequency of each subpopulation from live cell gate is shown (bottom). Data are pooled

from three experiments with 11/8 mice in each group. (e) Notch2 expression in myeloid cell subpopulations from BM of GFPþNotch2DMy mice. (f) Notch2

expression in Ly6Chi and Ly6Clo monocyte subpopulations isolated from BM. Littermate controls are shown for comparison. (g) Quantification of monocyte

subpopulations in mice with conditional deletion of Notch2 and expressing two alleles of LysMCre. Absolute number of cells per mg BM, per ml blood or per

spleen is shown (top). Relative frequency of each subpopulation from live cell gate is shown (bottom). Data are pooled from three experiments with 12/7 mice

in each group. (h) Correlation of Notch2þ Ly6Chi monocyte frequency with frequency of Ly6Clo monocytes. Frequency of Notch2þLy6Chi monocytes shows

strong positive correlation with Ly6Clo monocyte numbers (n¼ 28). (i) Quantification of monocyte subpopulations in Notch2DCD11c mice. Data are pooled from

two experiments with four mice in each group. (a,c,d,g,i) *Po0.05, **Po0.01, ***Po0.001; Student’s t-test. Error bars represent s.e.m.




Ly6Clo monocytes was strongly reduced, while the fraction ofrecovered Ly6Chi monocytes was significantly increased(Fig. 4c,d, Supplementary Fig. 6). Similar results were obtainedin experiments when peripheral Ly6Chi monocytes retrieved fromPB and spleen were used for transfer (Supplementary Fig. 7),which also ruled out development of Ly6Clo monocytes fromcontaminating BM precursors. Thus, Ly6Chi monocytes deficientfor Notch2 show impaired conversion into Ly6Clo monocytes,demonstrating regulation of monocyte cell fate by Notch2.

Dll1-Notch2 axis controls monocyte conversion in vitro. Notchreceptors are differentially engaged by Notch ligands, and Notch2is a preferred target of DLL1 (ref. 24). To define the Notchsignalling components regulating monocyte conversion, and toprovide proof-of-principle that Notch activation is sufficientto regulate this process, we established an in vitro culture systemto mimic the initial steps during monocyte conversion underdefined conditions. We sorted Ly6Chi monocytes from BM ofCx3cr1GFP/þ mice (Fig. 5a day 0) and cultured them in thepresence of immobilized recombinant DLL1 protein, or controlconditions (Fig. 5a and Supplementary Fig. 8). In culture,downregulation of Ly6C and upregulation of GFP occurred in allconditions over time, while cells remained uniformly CD115þ

(Fig. 5a, Supplementary Fig. 8b). However, conversion of Ly6Chi

into Ly6Clo monocyte-like cells, detected by upregulation ofCD43 and CD11c in MHC-IIlo/� cells, occurred to a significantlygreater extent on DLL1 than in control cultures (Fig. 5a,b).Furthermore, in gene expression profiling, cells cultured on DLL1showed significantly higher levels of Nr4a1 and Pou2f2 and sig-nificantly lower levels of Slfn5 compared with control culture(Fig. 5c), similar to a Ly6Clo monocyte phenotype3.

The effect was specific for DLL1, since culture of Ly6Chi

monocytes on another Notch ligand, JAG1, was much less effectivein generating Ly6Clo monocyte-like cells, as were controlconditions (Fig. 5d,e). Furthermore, DLL1-induced monocyteconversion was impaired by incubation with a g-secretaseinhibitor, N-(N-(3,5-difluorophenacetyl)-L-alanyl)-S-phenylgly-cine t-butyl ester (DAPT), which blocks the generation of theactive intracellular Notch domain37, thus indicating thatDLL1-induced Notch receptor cleavage is required for thisprocess (Fig. 6a,b). Importantly, DLL1-induced monocyteconversion was also severely impaired in Ly6Chi monocytes fromNotch2 conditional mutants when compared with controls(Fig. 6c,d). These results demonstrate that a specific Dll1-Notch2signalling axis controls conversion of Ly6Chi monocytes intoLy6Clo monocyte-like cells.

Dll1 is expressed in distinct endothelial niches. We wanted tocorroborate the specific function of Dll1 for Ly6Clo monocytedevelopment in vivo. In the adult mouse, Dll1 is selectivelyexpressed in vascular endothelium of arteries, but not veins orcapillaries, and in EC in the MZ of the spleen24,37. We firstcharacterized in more detail Dll1 expression in the two principlehaematopoietic compartments, BM and spleen, using geneticreporter mice or immunostaining. In Dll1þ /lacZ reporter mice, inwhich one allele of Dll1 has been replaced by lacZ, specificreporter staining was observed in the splenic MZ, but not in thecentral artery of the splenic follicle (Fig. 7a). Immunofluorescencestaining against CD31 and DLL1 and confocal microscopyrevealed DLL1 expression in EC of the MZ and confirmed itsabsence in the central artery of the follicle (Fig. 7b). Interestingly,DLL1 staining in Cx3cr1GFP/þ reporter mice demonstrated a

c

0.58 2.66

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Figure 3 | Ly6Clo monocytes show quantitative and phenotypical defects. (a) Intravital microscopy image showing GFPþ cells in microcirculation of

cremaster. Scale bar, 50mm. (b) Quantification of adherent (left) or rolling (right) monocytes per field of view in cremaster blood vessels at t¼0 or 60 min

after infusion of TNF-a. Data are pooled from two experiments (n¼ 6/7). ***Po0.001; two-way ANOVA with Bonferroni’s post-test. (c,d) GFPþNotch2DMy

(c) and Notch2DCD11c (d) mice develop an atypical CD11c�CD43�MHC-IIþ Ly6Clo population. Representative plots from Lin�CD11bþGFPþF4/

80loLy6Clo/� or Lin�CD11bþCX3CR1þF4/80loLy6Clo/� parental gates, respectively. (e) Representative flow cytometry plot showing CCR2 and CD11a

expression in Ly6Clo monocytes or atypical cells.




close spatial relationship between DLL1 expression and GFPþ

cell populations in the MZ, suggesting a potential niche function(Fig. 7c). Co-staining with Ly6C or CD43 identified bothmonocyte subsets within the CD31þ and DLL1þ MZ area,while large GFPþ macrophages reside in the borders of the MZ(Fig. 7d,e and Supplementary Fig. 9a,b). Furthermore, specificDll1-reporter staining in Dll1þ /lacZ mice was also observed in the

BM, which appeared in a reticular pattern in the diaphysal area ofthe BM cavity, suggesting a vascular pattern (Fig. 7f). Moreimportantly, the defect in Ly6Clo monocytes observed in Notch2conditional mutants was recapitulated in haploinsufficient Dll1mutant mice (Fig. 7g). This demonstrates a critical role for Dll1 inthe regulation of Ly6Clo monocytes in vivo, and emphasizes theimportance of a Dll1-Notch2 axis in the control of Ly6Clo

0.0

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a b

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Figure 4 | Notch2-deficient Ly6Chi monocytes show impaired conversion potential in vivo. Flow-cytometry based quantification of the fraction of

(a) apoptotic cells (AnnexinVþ7-AAD�) or (b) dead cells (7-AADþ) in monocyte subsets. Data are pooled from three-independent experiments

(n¼8/12). (c) Flow cytometry four days after adoptive transfer of BM Ly6Chi monocytes from control or Notch2-deficient CD45.2þGFPþ donors into

CD45.1þ congenic recipients. Transferred cells are shown in black and for comparison, recipient CD45.1þ (first row), CD45.1þCD11bþ (second row),

CD45.1þCD11bþ Ly6Chi (third row) cells or CD45.1þCD11bþLy6ChiF4/80lo monocytes (fourth and fifth rows) are depicted in blue. (d) Frequency of

donor-derived Ly6Clo monocytes (left) or Ly6Chi monocytes (right) in the CD11bþGFPþCD45.2þ gate after adoptive transfer of Ly6Chi monocytes. Data

are pooled from four-independent experiments (n¼ 5/7). (a,b,d) *Po0.05, **Po0.01, ***Po0.001; Student’s t-test. Error bars represent s.e.m.




monocyte development. Furthermore, these observations alsosuggest that distinct Dll1þ EC might form specialized niches forthe generation of Ly6Clo monocytes.

Endothelial Dll1 controls Ly6Clo monocyte development. Totest the hypothesis that endothelial Dll1 regulates Ly6Clo monocyte

development we employed an endothelial-specific and inducibledeletion strategy using the Cdh5(PAC)-CreERT2 strain, whichshows EC-specific Cre activity in peripheral vessels and the BMcavity16,38. We first confirmed pan-endothelial, but EC-specific,targeting by generating Cre-dependent lacZ-reporter mice(lacZiEC), which demonstrated inducible EC staining in arteries,veins and capillaries after tamoxifen treatment, but also

e

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3.2

Day 0

Figure 5 | Notch ligand DLL1 mediates monocyte conversion in vitro. (a,b) Kinetics of monocyte conversion in the presence of Notch ligand DLL1 in vitro.

Representative flow cytometry plots (a) and frequency (b) of Ly6Clo monocyte-like cells (CD11bþGFPþLy6Clo/�CD11cloCD43þMHC-IIlo/� ) calculated

from live CD11bþGFPþ cells are shown (n¼ 3). (c) Gene expression analysis in in vitro cultures from sorted BM Ly6Chi monocytes. Change relative to

expression of genes in control cultures is shown (n¼ 6). (d,e) DLL1 specifically induces conversion in vitro. Representative flow cytometry plot (d) and

frequency (e) of Ly6Clo monocyte-like cells (similar to Fig. 5a,b) are shown after culture of Ly6Chi monocytes alone or in the presence of control, JAG1 or

DLL1 ligands. (b,c,e) *Po0.05, **Po0.01, ***Po0.001; one-way ANOVA with Bonferroni’s multiple comparison test. Error bars represent s.e.m.




demonstrated Cre activity in splenic MZ EC and BM EC (Fig. 8a,Supplementary Fig. 9c). Employing a conditional allele of Dll1(ref. 23) we next generated endothelial-specific and inducibleDll1 mutant mice (Dll1iDEC) and confirmed Cre-dependentrecombination and deletion of the conditional Dll1 allele after apulse of tamoxifen (Supplementary Fig. 9d,e). In controlledexperiments (Supplementary Fig. 7f,g), endothelial deletion ofDll1 resulted in the selective reduction of Ly6Clo monocytes, whileLy6Chi monocytes and monocyte progenitors where unchangedcompared with control (Fig. 8b). These data demonstrate thatendothelial Dll1 regulates Ly6Clo monocytes in vivo.

Although our data clearly indicated the importance ofendothelial Dll1 for monocyte conversion, given the selectiveexpression of Dll1 in two distinct endothelial domains, arteriesand the haematopoietic compartment (BM and spleen), theidentity of the endothelial domain mediating monocyteconversion remained unclear and could not be addressed withour pan-endothelial deletion strategy. We, therefore, generatedmice with inducible, but arterial EC-specific deletion of Dll1, bycrossing the floxed allele of Dll1 to Bmx(PAC)-CreERT2 mice(Dll1iDaEC)39. We confirmed the arterial EC-specific Cre activityin a Cre-dependent lacZ-reporter strain (lacZiaEC), which

demonstrated specific EC staining in aorta, peripheral arteriesand central arteries of the splenic follicles, while MZ EC were nottargeted, thus providing a tool to address the contribution ofarterial EC to monocyte conversion (Supplementary Fig. 10a).We induced Dll1 deletion in arterial EC, which resulted inCre-dependent recombination of the conditional Dll1 allele(Supplementary Fig. 10b). In contrast to pan-endothelialdeletion of Dll1, arterial EC-specific deletion of Dll1 did notaffect relative or absolute numbers of Ly6Clo monocytes (Fig. 8c),suggesting that Dll1 expressed in endothelial niches in the MZ orBM, and not in arterial endothelium, regulates monocyteconversion.

To further investigate the specificity of Dll1 effects on Ly6Clo

monocytes we mated the pan-endothelial and inducibleCre-transgenic strain to conditional alleles of the related Notchligand Dll4 (refs 38,40). In contrast to deletion of Dll1, deletion ofDll4 did not lead to alterations in Ly6Clo monocytes, or any othermyeloid subset (Fig. 8d), which demonstrates specific effects ofDll1 in the regulation of Ly6Clo monocytes.

To provide proof-of-principle that EC populations from thehaematopoietic compartment regulate monocyte conversionwe established an in vitro co-culture system. CD144þ ECs

GFP +N2 ΔMy

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Ctr

l

Figure 6 | Notch2 signalling is required for monocyte conversion in vitro. (a,b) Inhibition of Notch signalling in sorted Ly6Chi monocytes using a

g-secretase inhibitor (DAPT) impairs conversion in vitro. Representative flow cytometry plot from live CD11bþGFPþ cells (a) and relative frequency (b) of

Ly6Clo monocyte-like cells (CD11bþGFPþLy6Clo/�CD11cloCD43þMHC-IIlo/� ) are shown. (c,d) Ly6Chi monocytes from GFPþNotch2DMy mice show

impaired conversion. Representative flow cytometry plot (c) and relative frequency (d) of Ly6Clo monocyte-like cells are shown (similar to Fig. 6a,b).

(b,d) n¼ 3, *Po0.05, **Po0.01, ***Po0.001; one-way ANOVA with Bonferroni’s Multiple comparison test. Error bars represent s.e.m.




were sorted from splenic tissue digests and cultured withLy6Chi monocytes from Cx3cr1GFP/þ reporter mice. We alsoconfirmed Dll1 expression in this EC population (Fig. 8e).Compared with monocytes cultured alone, conversion of Ly6Chi

monocytes into Ly6Clo monocyte-like cells was significantly

increased on splenic EC, an effect that persisted over time(Fig. 8f,g). Altogether, these results suggest that distinctendothelial niches in the haematopoietic compartments regulateconversion of Ly6Chi monocytes into Ly6Clo monocytes via theDll1-Notch2 axis.

a

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Figure 7 | Dll1 deficiency impairs Ly6Clo monocyte development in vivo. (a) MZ-specific b-galactosidase activity in splenic follicle of Dll1þ /lacZ mice

(left), but not in wt control (right). Scale bars, 50mm. (b) Immunostaining and confocal microscopy of splenic follicle MZ demonstrating DLL1 expression in

CD31þ EC. Scale bar, 20mm. (c) Immunostaining and confocal microscopy showing GFPþ cells in association with DLL1þ structures in splenic follicle MZ.

Scale bar, 50mm. (d,e) Immunostaining and confocal microscopy showing GFPþLy6Cþ (d) or GFPþCD43þ (e) cells in CD31þ areas in splenic follicle

MZ. Large GFPþ cells bordering MZ are Ly6C or CD43 negative. Scale bar, 50mm. (f) Dll1 expression in BM of femur diaphysis indicated by specific

b-galactosidase staining in Dll1þ /lacZ mice; scale bars, 100 mm. (a–f) representative of more than two experiments. (g) Relative numbers of myeloid cell

populations by flow cytometry in Dll1þ /lacZ mice or wt littermate controls. Data are pooled from four-independent experiments (n¼ 11/12). *Po0.05,

**Po0.01, ***Po0.001; Student’s t-test. Error bars represent s.e.m.




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f

Figure 8 | Endothelial deletion of Dll1 impairs Ly6Clo monocyte development in vivo. (a) Inducible and endothelial-specific Cre-reporter mice (lacZiEC)

showing specific b-galactosidase staining in splenic MZ (left) and BM (right) of the femur diaphysis after tamoxifen treatment; scale bars, 50 mm.

(b) Absolute and relative numbers of myeloid cell populations by flow cytometry after pan-endothelial deletion of Dll1 compared with littermate controls.

Data are pooled from four experiments (n¼ 8). (c) Absolute and relative numbers of myeloid cell populations by flow cytometry after arterial-specific,

endothelial deletion of Dll1 compared with littermate controls. Data are pooled from two experiments (n¼ 5/6). (d) Absolute and relative numbers of

myeloid cell populations by flow cytometry after endothelial-specific deletion of Dll4 compared with littermate controls. Data are pooled from two

experiments (n¼ 5). (e) Quantitative reverse transcription–PCR analysis of Dll1 expression in sorted splenic monocytes (CD11bþGFPþLy6ChiCD144� )

and ECs (CD11b�GFP�CD144þ ). Data are pooled from three-independent experiments (n¼6). (f,g) Co-culture of splenic CD144þ ECs with

Ly6Chi monocytes. Representative flow cytometry plot from GFPþCD11bþ gate (f) and frequency of Ly6Clo monocyte-like cells (g) are shown (n¼ 3).

*Po0.05, **Po0.01, ***Po0.001; Student’s t-test (b–e) or one-way ANOVA with Bonferroni’s multiple comparison test (g). (b–e,g) Error bars

represent s.e.m.




DiscussionWe here define the molecular and cellular context that regulatesmonocyte conversion. Taken together, our results show thatNotch2 regulates conversion of Ly6Chi monocytes into Ly6Clo

monocytes, thereby regulating a specific developmental stepin monocyte cell fate under steady-state conditions. Thisprocess is controlled specifically by Notch ligand Dll1 expressedby a population of EC that constitute distinct vascular niches inthe BM and spleen. Thus, blood vessels regulate monocyteconversion, as a form of developmental cell fate regulation.

The origin and regulation of Ly6Clo monocytes is still poorlyunderstood. Recent findings have suggested that Ly6Clo

monocytes develop from Ly6Chi monocytes. Adoptive transferof cMoP leads to the sequential appearance of Ly6Chi monocytesfollowed by Ly6Clo monocytes3. Furthermore, Jung andcolleagues have provided direct evidence that Ly6Clo monocytesderive from Ly6Chi monocytes by adoptive transferexperiments7,14. Our experiments demonstrating monocyteconversion with isolated Ly6Chi monocytes in vivo, andrecapitulation of it in vitro, clearly support the notion ofmonocyte conversion as a mechanism regulating developmentof Ly6Clo monocytes. However, while our data provideevidence for and insights into the mechanism of monocyteconversion, our findings do not exclude the existence ofadditional mechanisms to generate Ly6Clo monocytes, forexample from progenitor cells, as has been recently suggested41.

So far, specific molecular or cellular regulators of monocyteconversion have remained elusive, and it has been speculated thatmonocyte conversion occurs spontaneously in the circulation2,14.Our data demonstrating a requirement for the Dll1-Notch2signalling axis for the conversion of Ly6Chi monocytes intoLy6Clo monocytes not only provides clear evidence for theregulated nature of monocyte conversion, but also shows that thisprocess is under control of blood vessels through Notch ligandDll1. The fact that conditional deletion of Dll4 has no impact onLy6Clo monocytes further emphasizes the specific nature of Dll1actions. Although the precise location of monocyte conversionremains unknown, our data clearly demonstrate the existence,and functional importance, of distinct Dll1-expressing vascularniches in BM and spleen. This suggests that monocyte conversionhappens, or is at least initiated, in these vascular niches understeady-state conditions. This is further supported by the fact thatco-cultured EC from these niches promote monocyte conversion,while our in vivo data from mice with arterial-specific Dll1deletion exclude the participation of arterial ECs in this process.However, given the dynamic nature of endothelial responses, it isconceivable that in certain situations, such as inflammation, thelocation, composition and extent of vascular niches might change,which, in turn, might influence monocyte conversion rates.

Recently, the instructive function of the vascular niche forself-renewal and regenerative capacity of HSCs has been defined,which, in part, is mediated by endothelial-specific expression ofNotch ligand Jag1 (refs 20,21). Our finding that monocyteconversion is regulated by ECs extends the spectrum ofniche regulation towards more committed steps of myeloid celldevelopment and further supports the importance of the vascularniche in regulating cell fate. On the other hand, the finding thatmonocyte conversion is specifically regulated by vascular Dll1underlines the ligand-specific nature of Notch signalling events indifferent locations16.

Our data also suggest that Notch2 expressed in monocytes isdirectly involved in regulation of Ly6Clo monocyte cell fate inresponse to Dll1. Several lines of evidence support thisconclusion. First, Notch2 was required for monocyte conversionin experiments using isolated Ly6Chi monocytes in vivo andin vitro. Second, when Notch2 was targeted on Ly6Chi monocytes

in vivo we found that the extent of Notch2 loss-of-function inLy6Chi monocytes is related to the loss of Ly6Clo monocytes,while Ly6Chi monocyte numbers are not affected by Notch2deletion. On the other hand, targeting of Notch2 in Ly6Clo

monocytes also resulted in selective deletion of Ly6Clo

monocytes. Together, this demonstrates a requirement formonocyte Notch2 in the generation, as well as maintenance ofLy6Clo monocytes. Finally, Notch2 loss-of-function lead to theappearance of an atypical monocyte population negative forCD11c and CD43 but expressing high levels of MHC-II andCCR2. These findings suggest a model in which Notch2regulates Ly6Clo monocyte cell fate: active Notch2 signallingmediates conversion into Ly6Clo monocytes, while defectiveNotch2 signalling leads to MHC-IIhi atypical monocytes. Whilethe role and relevance of this atypical monocyte populationobserved in conditional mutants is currently unclear, it isimportant to note that, using different gating strategies andexperimental approaches, subpopulations of MHC-II-expressingmonocytes have recently been described, which acquire antigenfor carriage to lymph nodes36.

Currently, the molecular effectors of Notch2 in monocytes areunknown. Interestingly, the transcription factor Nr4a1, an orphannuclear receptor, regulates the survival of Ly6Clo monocytes13.Although a role of Nr4a1 in monocyte conversion has not beeninvestigated, mice with general or BM-restricted inactivation ofNr4a1 showed reduced Ly6Clo monocytes numbers and increasedrates of apoptosis. Although we did not observe a cell deathphenotype in Notch2 mutant mice, we found DLL1-dependentregulation of Nr4a1 in vitro. Clearly, the molecular regulation ofmonocyte conversion requires further study.

Our study also describes the first steps towards an approach torecapitulate monocyte cell fate ex vivo. This might provide asetting to study and understand the molecular events drivingmonocyte conversion under defined conditions.

MethodsMice. Mouse strains used in the study are listed in Supplementary Table 2.Cx3cr1GFP/þ mice28 (kindly provided by Steffen Jung), LysMCre mice32, Dll1þ /lacZ

mice42 (kindly provided by Achim Gossler), Notch1lox/lox and Notch2lox/lox mice17,Cdh5(PAC)-CreERT2 mice43, Bmx(PAC)-CreERT2 mice44, Dll1lox/lox mice23,Dll4lox/lox mice45, N2DCD11c mice46, N1N2DCD11c mice47, Nr4a1-GFP mice29,LysM-eGFP mice33, LysMCreRosaYFP mice48 have been described. Gt(ROSA)26Sormice carrying Cre-inducible lacZ alleles were obtained from The JacksonLaboratories, B6.SJL-PtprcaPepcb/BoyJ (CD45.1þ ) mice from Charles River. Micewere housed under specific pathogen free conditions in the animal facility ofHannover Medical School unless otherwise indicated. Nr4a1-GFP, LysM-eGFP andLysMCreRosaYFP mice were housed in IPEC, Munich, Germany; N2DCD11c mice inWUSTL, St Louis, MO, USA; N1N2DCD11c mice in Tokushima University,Tokushima, Japan and Cdh5(PAC)-CreERT2 Dll4lox/lox mice in MPI Munster,Germany. All experiments were performed with 8–12-weeks-old mice and age andsex matched littermate controls with approval of the local animal welfare boards(LAVES Lower Saxony, Animal Studies Committee at Washington University in StLouis, Animal Research Committee of Tokushima University, North RhineWestphalia Animal Ethics Committee and Local Animal Committee of DistrictGovernment of Upper Bavaria).

Tissue and cell preparation. For single cell suspension mice were killed andspleen, BM and blood were collected. Erythrocytes were removed by red blood celllysis buffer (Biolegend) or by density centrifugation using Histopaque 1083 (Sigma-Aldrich). After extensive washing cells were resuspended in PBS containing 10% FCSand 2 mM EDTA kept on ice, stained and used for flow cytometry or for sorting.

Flow cytometry and cell sorting. Non-specific binding of antibodies byFc-receptors was blocked with anti-mouse CD16/CD32 (TruStain fcX fromBiolegend) in single cell suspensions prepared from spleen, PB or BM. Aftersubsequent washing step cells were labelled with primary and secondary antibodiesor streptavidin-fluorochrome conjugates (Supplementary Table 3) and used forflow cytometry analysis (LSR-II, BD Biosciences) or sorting (FACSAria; BDBiosciences or MoFlo XDP; Beckman Coulter). Data were analysed by FlowJosoftware (Treestar). Initially cells were identified based on forward scatter (FSC)and side scatter (SSC) characteristics. After exclusion of doublets (on the basis of




SSC-W, SSC-A), relative frequency of each subpopulation from live cell gate orabsolute number of each subset (calculated from live cell gate and normalized onmg BM, ml PB or spleen) were determined and are shown in the graphs asmean±s.e.m., unless otherwise stated.

In vitro conversion studies. Ninety-six-well flat bottom plates were coated at roomtemperature for 3 h with IgG-Fc, JAG1-Fc or DLL1-Fc ligands (all from R&D)reconstituted in PBS. Sorted BM Ly6Chi monocytes were cultured in coated plates inthe presence of M-CSF (10 ng ml� 1), thrombopoietin (TPO, 20 ng ml� 1), stem cellfactor (SCF, 10 ng ml� 1), insulin-like growth factor (IGF)-II (20 ng ml� 1), fibroblastgrowth factor (FGF)-I (10 ng ml� 1) (all from Peprotech) and Heparin (25 U ml� 1)at 37 �C for 24 or 48 h. In experiments where the effect of Notch inhibition onconversion process was assessed, 6mM of g-secretase inhibitor, DAPT or dime-thylsulphoxide (DMSO) was applied to the cells prior to and 24 h after culture. Inseparate experiments BM Ly6Chi monocytes were co-cultured with sorted splenicCD144þGFP�CD11b� EC. One or 2 days after culture, cells were harvested,stained and subjected to flow cytometry. Frequency of Ly6Clo monocyte-like cells(CD11bþGFPþLy6Clo/�CD11cloMHC-IIlo/�CD43þ ) in total live CD11bþ

GFPþ cells served as an indicator of conversion efficiency and is shown in thegraphs.

Adoptive cell transfer experiments. CD11bþLy6ChiGFPþ monocytes weresorted from BM and injected into CD45.1þ recipients intravenously (i.v.). Fourdays after transfer spleen, PB and BM were collected and single cell suspension wasprepared. After blocking of Fc receptors using anti-mouse CD16/CD32 (TruStainfcX from Biolegend) cells were labelled with biotin-conjugated anti-CD45.1 anti-body, anti-biotin magnetic beads and enriched on LD columns (Miltenyi Biotec)according to the manufacturer’s instructions. CD45.1 negative fraction was col-lected, stained and analysed by flow cytometry. Ly6Clo monocytes (CD11bþ

GFPþLy6Clo/�F4/80loCD11cloMHC-IIlo/� cells) were quantified in spleen, BMand PB as relative frequency of total donor derived CD45.2þCD11bþGFPþ cells.

Human monocyte isolation. Human monocytes were isolated from the blood ofhealthy individuals as approved by the ethical committee of Hannover MedicalSchool. Written consent was obtained before blood collection. Cells were purifiedusing CD16þ monocyte isolation kit (Miltenyi Biotec) according to themanufacturer’s instruction. CD14þ monocytes were retrieved in subsequentpurification step from CD16neg fraction using CD14 microbeads.

Immunohistochemistry and immunofluorescence. Immunohistochemistry,b-galactosidase and immunofluorescence staining in mice were performed withmodifications from previous descriptions37,49. Mice were euthanized; tibiae, spleen,heart, aorta and muscles were isolated and fixed in 4% paraformaldehyde (PFA).Bones were decalcified in 0.5 M EDTA solution at 4 �C for 48 h, cryopreserved insucrose and embedded in Tissue-tek O.C.T. compound (Sakura, Germany). All theother organs were cryoprotected in sucrose and embedded in Tissue-tek O.C.T.compound without decalcification procedure. b-galactosidase staining wasperformed at 37 �C on PFA fixed tissues. Slides were counterstained with eosin,mounted in mounting medium and analysed with Olympus IX71 microscope.For immunofluorescence and confocal laser scanning microscopy tissue sectionswere stained using anti-DLL1 (Biolegend), anti-CD31, anti-CD43 (both fromBD Biosciences, Germany), anti-Ly6C (Biolegend) and appropriate fluorescence-conjugated secondary antibodies (Supplementary Table 3). 4,6-Diamidino-2-phenylindole (Invitrogen, Germany) was used for counterstaining of nuclei andslides were mounted in DAKO fluorescence mounting medium (Dako, Denmark).Images were acquired using Leica TCS SP2 AOBS (Leica Microsystems, Germany)confocal microscope or Zeiss Observer Z1 fluorescence microscope (Zeiss,Germany), respectively.

Intravital microscopy. To visualize monocytes in the microcirculation thecremaster muscle of male GFPþ ctrl and GFPþN2DMy mice was exposed andtransient and adhesive interactions were recorded. To this end an Olympus BX51microscope equipped with a Hamamatsu 9100-02 EMCCD camera (HamamatsuPhotonics) and a � 40 water-dipping objective was employed. In each cremaster10 fields of view were recorded for 30 s and the number of adherent cells and therolling flux (rolling monocytes passing a perpendicular line placed across theobserved vessel) from each field were quantified. Subsequent to recordings atbaseline, mice were injected via a jugular vein catheter with a single dose of TNF-a(250 ng) and recordings were repeated after 60 min.

Quantitative real-time PCR analysis. Splenic ECs, as well as splenic or BMmonocytes were isolated by cell sorting and total RNA was purified usingNucleospin RNA II kit (Macherey Nagel). After purity and quality check, RNA wastranscribed into complementary DNA employing complementary DNA synthesiskit (Invitrogen) according to the manufacturer’s instructions. Quantitativereal-time PCR was performed using specific primers (Supplementary Table 4) andFastStart Essential DNA Green Master on a LightCycler 96 system from

Roche according to the manufacturer’s instructions, for normalization. Expressionof each specific gene was normalized to expression of Rps9 and calculated by thecomparative CT (2�DDCT) method50.

In vivo targeting of EC and PCR analysis. Five to 6-weeks-old mice expressingEC-specific inducible Cre recombinase and Cre-negative littermate controls weretreated with 500 mg tamoxifen for 5 consecutive days intraperitoneally (i.p.). Micewere killed after 4 weeks. Efficiency of Cre-dependent gene deletion was monitoredby PCR and agarose gel-electrophoresis as described23.

Statistical analysis. Results are expressed as mean±s.e.m.. N numbers arebiological replicates of experiments performed at least three times unless otherwiseindicated. Significance of differences was calculated using unpaired, two-tailedStudent’s t-test with confidence interval of 95%. For comparison of multipleexperimental groups one-way analysis of variance (ANOVA) was used andBonferroni’s multiple-comparison test was performed when the overall P valuewas o0.05. Data from intravital microscopy were analysed using two-wayANOVA and Bonferroni’s post-test. P values of less than 0.05 were considered tobe significant.

Data availability. The authors declare that all the relevant data are available uponrequest.

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AcknowledgementsWe thank Sylvana Henschen and Stefan Sablotny for excellent technical assistance;Achim Gossler for Dll1þ /lacZ mice; the Central Animal Facility, Research Core FacilityCell Sorting, Research Core unit Laser Microscopy of Hannover Medical School forexcellent support. This research has been funded by grants from DFG (Li948-5/1), BMBF(01GU1105B), IFB-Tx (01EO1302) and GIF (1089) to F.P.L. and SFB1054-B04 to C.W.

Author contributionsJ.G., R.G., J.J., O.S., J.D., C.G.B., K.K., A.L., T.K., S.K.R., C.I., R.H., L.C.N. did theexperiments; J.G., R.G., J.J., O.S., J.D., C.G.B., C.I., K.Y., F.P.L. designed and analysedexperiments; J.G., F.R., L.J.S., U.Z.S., J.B., H.H., R.H.A., C.W., K.M.M., K.Y., F.P.L. wroteand edited the manuscript; F.P.L. conceived of the research and directed the study.

Additional informationSupplementary Information accompanies this paper at http://www.nature.com/naturecommunications

Competing financial interests: The authors declare no competing financial interests.

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How to cite this article: Gamrekelashvili, J. et al. Regulation of monocyte cellfate by blood vessels mediated by Notch signalling. Nat. Commun. 7:12597doi: 10.1038/ncomms12597 (2016).

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