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Regulation of angiogenesis by a non-canonical Wnt–

LETTER

doi:10.1038/nature10085

Regulation of angiogenesis by a non-canonical Wnt–Flt1pathway in myeloid cells

James A.Stefater III 1,2,Ian Lewkowich 3,Sujata Rao 1,2,Giovanni Mariggi 4,April C.Carpenter 1,2,Adam R.Burr 5,Jieqing Fan 1,2,Rieko Ajima 6,Jeffery D.Molkentin 5,7,Bart O.Williams 8,Marsha Wills-Karp 3,Jeffrey W.Pollard 9,Terry Yamaguchi 6,Napoleone Ferrara 10,Holger Gerhardt 4,11&Richard http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.htmlng 1,2

Regulation of angiogenesis by a non-canonical Wnt–

Myeloid cells are a feature of most tissues.Here we show that during development,retinal myeloid cells (RMCs)produce Wnt ligands to regulate blood vessel branching.In the mouse retina,where angio-genesis occurs postnatally 1,somatic deletion in RMCs of the Wnt ligand transporter Wntless 2,3results in increased angiogenesis in the deeper layers.We also show that mutation of Wnt5a and Wnt11results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.htmling cultured myeloid-like cells and RMC somatic deletion of Flt1,we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1,a naturally occurring inhibitor of vascular endothelial growth factor (VEGF)4–6.These findings indicate that resident myeloid cells can use a non-canonical,Wnt–Flt1pathway to sup-press angiogenic branching.

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Myeloid cells have a wide array of biological activities that include immune activation,arteriogenesis 7,and regulation of salt balance and blood pressure 8.Myeloid cells also regulate vascularity.Tumour-associated macrophages influence the growth of blood vessels 9in part because they are a source of VEGFA 10.Myeloid cells also promote angiogenic branching 11and anastamosis 12.Depending on the context,myeloid cells can be either anti-angiogenic 13or pro-angiogenic 14.Here we show that RMCs suppress retinal angiogenesis via a Wnt–Flt1pathway (Supplementary Fig.1).

Regulation of angiogenesis by a non-canonical Wnt–

Retinal angiogenesis begins on the day of birth in the mouse with the formation of a superficial vascular plexus (Fig.1a)that lies within the ganglion cell layer 15.After formation of this superficial plexus by post-partum day 7(P7),angiogenic sprouts descend vertically through the retinal layers from P8to P14(Fig.1a).At the outer edge of the inner nuclear layer (INL)the vertical angiogenic sprouts turn and simulta-neously branch to form a deep vascular plexus (Fig.1a).Using antibodies to the vascular endothelial cell (VEC)marker endomucin and to the green fluorescent protein (GFP)of the c-fms–EGFP (also known as Tg(Csf1r-EGFP)1Hume )transgene that marks RMCs,we show that myeloid cells have a unique spatial relationship with angiogenic tip cells.At the point of turning and branching in the outer INL,myeloid cells and angiogenic sprouts are in close contact (Supplementary Fig.2a).This was confirmed by labelling with isolectin and F4/80(F4/80is also known as Emr1;Supplementary Fig.2b).We then took advantage of high-intensity isolectin labelling of VECs and RMCs and performed a three-dimensional reconstruction with false colouring to illustrate the overall topology of the angiogenic tip cell–RMC interaction (Fig.1b).This showed close contact between the two cell types throughout turn-and-branch angiogenesis in the deep retinal layer.Furthermore,after turning,angiogenic tip cells extend within the plane of the deep retinal layer and remain RMC-associated (Fig.1c).In further defining RMCs,

1

The Visual Systems Group,Divisions of Pediatric Ophthalmology and Developmental Biology,Cincinnati Children’s Hospital Medical Center,Cincinnati,Ohio 45229,USA.2Department of Ophthalmology,University of Cincinnati,Cincinnati,Ohio 45229,USA.3Division of Immunobiology,Cincinnati Children’s Hospital Medical Center,Cincinnati,Ohio 45229,USA.4Vascular Biology Laboratory,London Research Institute,Cancer Research UK,London WC23PX,UK.5Division of Molecular Cardiovascular Biology,Cincinnati Children’s Hospital Medical Center,University of Cincinnati,Ohio 45229,USA.6

Cancer and Developmental Biology Laboratory,National Cancer Institute,Frederick,Maryland 21701,USA.7Howard Hughes Medical Institute,Cincinnati Children’s Hospital Medical Center,University of Cincinnati,Ohio 45229,USA.8Center for Skeletal Disease Research,Van Andel Research Institute,333Bostwick NE,Grand Rapids,Michigan 49503,USA.9Albert Einstein College of Medicine of Yeshiva University,Jack and Pearl Resnick Campus,1300Morris Park Avenue,Bronx,New York 10461,USA.10Genentech Inc.,1DNA Way,South San Francisco,California 94080,USA.11Consultant Group Leader,Vascular Patterning Laboratory,Vesalius Research Center,VIB,Campus Gasthuisberg,B-3000Leuven,

Belgium.

Isolectin vascular endothelial cells Isolectin false-colour myeloid cells

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Figure 1|RMCs interact with VECs and express Wnt components.

a ,Schematic of retina at postnatal days (P)2,6,10,and 18.RMCs interacting with descending vertical sprouts are labelled with red arrowheads.Adapted from ref.1.

b ,Isolectin-labelled three-dimensional reconstruction of vertical angiogeni

c sprouts (green)an

d RMCs (false-colour red).c ,As in b but a two-dimensional imag

e in the deep vascular layer.Scale bars,5m m.d ,e ,Flow cytometry o

f deep layer RMCs based on surface markers.7-AAD,

7-aminoactinomycin D.f ,PCR for Wnt pathway components on flow-sorted RMCs.Red arrowheads indicate expected sizes.Act,b -actin.

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we showed that CD204(also known as Msr1)was unique to RMCs with amoeboid morphology in the superficial retinal layer (Supplementary Fig.2c,d).By contrast,deep retinal layer RMCs with extended morpho-logy were CD204-negative (Supplementary Fig.2c,e).Both layers of RMCs expressed Iba1(ref.16)but at different levels (Supplementary Fig.2c–e).This information allowed us to sort distinct populations of super-ficial (CD11b 1,F4/801,CD2041)and deep layer (CD11b 1,F4/801,CD204-)RMCs by flow cytometry (Fig.1d,e).

On the basis of previous work showing vascular regulation by myeloid Wnt ligands 17we proposed that RMCs might use Wnt ligands to regulate retinal angiogenesis.First,we examined the expression of Wnt ligands and receptors in superficial and deep RMCs isolated by flow cytometry.PCR with reverse transcription (RT–PCR)analysis of the deep RMC population showed expression of Wnt5a,Wnt6and Wnt11,Fzd7and Fzd8as well as the co-receptor Lrp5(Fig.1f).With the exception of Wnt5b,which was inconsistent,no other Wnt ligand was detected in deep RMCs.Superficial RMCs expressed similar Wnt and Fzd proteins (Fig.1f),but also expressed Wnt2b,3and 3a (data not shown).

The challenge of genetic analysis when RMCs express many Wnt ligands was addressed by the generation of a loxP -flanked conditional

allele for the essential Wnt ligand transporter Wls 2,3.Both superficial and deep RMCs expressed Wls (Fig.1f).Wls was deleted using the myeloid cre driver cfms–icre 18,which we confirmed was functional in RMCs (Supplementary Fig.3a–c).To analyse retinal vasculature,we imaged superficial and deep retinal vessels at P18(Fig.2a).Quantification of vessel branch points showed that cfms–icre alone had no effect (Fig.3b).Furthermore,compared with control Wls fl/1mice,Wls fl/1;cfms–icre animals had a normal superficial vascular plexus (Fig.2a c).By contrast,the deep vascular layer (Fig.2a,c)showed an overgrowth.Interestingly,no further enhancement of vas-cular overgrowth was apparent when the myeloid Wls deletion was homozygous as in Wls 2/fl ;cfms–icre mice (data not shown).Because myeloid cells are positioned below descending vessels at P10,we assessed vessel branching at the base of these sprouts.Wls fl/1;cfms–icre mice showed reduced simple turning (no branches)and single branching,but significantly more multi-branch events (Fig.2d).As a weighted mean,the branch index was 2.0in control and 3.2in the mutant (P ,0.0001).

The higher vascular density of the deep layer in Wls fl/1;cfms–icre mice could reflect enhanced angiogenesis or defective remodelling.

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

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Figure 2|RMC Wls is required for suppression of deep angiogenic

branching.a ,Isolectin labelling of superficial and deep retinal vasculature in Wls fl/1and Wls fl/1;cfms–icre mice.Scale bars,50m m.b ,c ,P18vessel branch points in labelled genotypes.n 54(b ),n 58(c ).d ,Branches emanating from the base of vertical sprouts in the P10deep vascular layer.n 58.b –d used

Student’s t -test (two-tailed).e ,Time-course of deep layer branches in indicated genotypes.Shading shows when angiogenesis (green)and remodelling (red)predominate.One-way ANOVA showed P 50.0021.n 54for each point.Error bars are s.e.m.,NS,not significant.

RESEARCH LETTER

2|N A T U R E |V O L 000|00M O N T H 2011

assess this,we counted deep layer branch points in control (Wls fl/1)and experimental (Wls fl/1;cfms–icre )mice over a time-course from P6to P18(Fig.2e).Between P14and P18when remodelling predominates (Fig.2e,red zone)control and mutant graph slopes were nearly ident-ical.By contrast,between P6and P14when vessel growth predomi-nates (Fig.2e,green zone),the slope of the graph is greater in the mutant.There was no difference between control and mutant animals in the number of vertical sprouts connecting superficial and deep layers (Supplementary Fig.3d).Combined,the data in Fig.2are con-sistent with a model in which myeloid Wnt ligands suppress branch formation as angiogenic sprouts make contact with deep layer RMCs (Fig.1b,c).These data were corroborated by the vascular overgrowth phenotype of Wnt5a (ref.19)and Wnt11(ref.20)heterozygotes (Supplementary Figs 3e,f and 4).

Wnt5a and Wnt11are most often associated with non-canonical Wnt signalling 21.We thus investigated the possibility that Wnt-dependent suppression of deep retinal angiogenesis was a non-canonical response.Wnt3a,but not Wnt5a,elicited a canonical response in SuperTopflash (STF)reporter cells (Supplementary Fig.5a).Some non-canonical Wnt pathways activate a Ca 21flux 21.To determine whether myeloid Wnt proteins elicited a Ca 21flux,we added Wnt5a to myeloid-like RAW264.7cells and measured Ca 21-dependent Fura-2dye emission.Recombinant Wnt5a increased intracellular Ca 21compared to untreated controls (Fig.3a and Supplementary Fig.5b,c).Furthermore,to assess the requirement for Wls in Wnt ligand secretion,we exposed RAW264.7cells to medium from Wls fl/fl mouse embryonic fibroblasts (MEFs)transfected with Wnt5a or Wnt5a and cre recombinase plasmids.RAW264.7cells had increased Ca 21flux in response to Wnt5a -transfected MEF medium relative to Wnt5a -and cre -transfected medium (Fig.3b).This validates the role of Wls in secretion of http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.htmlbined,these data show that one myeloid Wnt,Wnt5a,does not stimulate a canonical Wnt response,but can elicit a Ca 21flux characteristic of some non-canonical Wnt responses.

In recent work,it has been shown that Wnt5a loss-of-function can rescue the defects associated with deletion of the canonical Wnt pathway co-receptors Lrp5and Lrp6(ref.22).This finding has suggested that Lrp5/6deletion actually represents a non-canonical pathway gain-of-function and that in deleting the non-canonical ligand Wnt5a,there is a re-balancing of pathway activity.This hypothesis is supported by bio-chemical analysis showing that Wnt5a can bind Lrp6,but does not elicit the phosphorylation required for canonical signalling 22.Canonical,non-canonical reciprocal pathway inhibition has also been demon-strated 23.These findings argue that a deletion of Lrp5/6can define whether a Wnt signalling pathway is canonical or non-canonical.If it is canonical,an Lrp5/6deletion will give the same phenotype as ligand deletion.By contrast,if the signalling pathway is non-canonical,the consequence of Lrp5/6deletion would be opposite to that of ligand mutation.Thus,to determine whether the RMC response was canonical or non-canonical,we generated a cfms–icre somatic mutant of the Lrp5coreceptor that is expressed in RMCs (Fig.1f).Because the result was significantly diminished deep vascular layer density in somatic homo-zygotes (Fig.3c–f),a response opposite to ligand deletion,this provides in vivo evidence that the Wnt response is non-canonical.Even though the superficial vascular layer was slightly deficient in somatic homozy-gotes (Fig.3e)this was not the reason for reduced density in the deep vascular layer as the descending sprout number was unchanged (Fig.3f).The VEGF receptor Flt1(also known as VEGFR1)can suppress angiogenesis because it has limited signalling capacity,a higher affinity for VEGF than Flk1(also known as VEGFR2)and can sequester VEGF 5,6.Alternative splicing produces both membrane-tethered and soluble forms 5.Flt1is known to contribute to corneal avascularity 4as well as the selection of angiogenic tip cells when expressed regionally in an existing vessel 24.Flt1is also known to be expressed in some myeloid populations 6.Because angiogenesis in the deep retinal layers is VEGF-dependent 25,Flt1was a good candidate to mediate Wnt-dependent angiogenic suppression by deep

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

RMCs.

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Figure 3|Angiogenic suppression by RMCs is a non-canonical Wnt

response.a ,b ,Intracellular Ca 21in RAW264.7cells treated with Wnt5a (a )or supernatant from Wls fl/fl MEFs expressing Wnt5a or Wnt5a and cre (b ).One Way ANOVA showed P #0.0001for both.c ,d ,Isolectin labelling of superficial and deep retinal vasculature in Lrp5fl/1(c )and Lrp fl/fl ;cfms–icre (d )mice.

50m m scale bars.e ,P18vessel branch points in labelled genotypes.f ,Vertical vessels connecting to the deep layer in labelled genotypes.e ,f ,Used one-way ANOVA with Tukey’s post-hoc.n $8per genotype.Errors are s.e.m.,NS,not significant.

LETTER RESEARCH

00M O N T H 2011|V O L 000|N A T U R E |3

We thus determined whether Flt1was expressed in P12RMCs by performing end-point RT–PCR on flow-sorted cells using primers that detected both transcripts.This showed that deep RMCs expressed Flt1(Fig.4a).By contrast,superficial RMCs did not (Fig.4a).We also showed that Iba1-labelled RMCs positioned at the outer edge of the INL labelled with anti Flt1antibodies (Fig.4b).We then generated an RMC Flt1loss-of-function using cfms–icre and the Flt1fl conditional allele 26.Quantification of retinal vascular branch points in Flt1fl/1;cfms–icre mice at P18showed that the superficial layer was unaffected but that the deep layer showed an increase in density (Fig.4c,d).As with Wls somatic mutants,conditional homozygosity for Flt1(Flt1fl/fl ;cfms–icre )did not give a further significant increase in vascular density (data not shown).Furthermore,the number of deep RMCs was unchanged (Supplementary Fig.2g).

These data showed that conditional deletion of Flt1produced an enhancement of angiogenesis similar to that observed with conditional

deletion of Wls .One implication was that Wls and Flt1might function in the same angiogenesis suppression pathway.To test the possibility that Flt1might be regulated by Wnt ligands,we used myeloid-like RAW264.7cells that,like RMCs,express Fzd7and Fzd8(Fig.4e).When stimulated with the ‘canonical’ligand Wnt3a,RAW264.7did not change the level of Flt1transcript according to quantitative PCR (Fig.4f).However,stimulation with Wnt5a produced a threefold increase in the level of both Flt1isoform transcripts (Fig.4f).Furthermore,an enzyme-linked immunosorbent assay (ELISA)showed that there was an increased level of soluble Flt1in conditioned media from Wnt5a-stimulated RAW264.7cells (Fig.4g).Wnt3a stimulation again had no effect (Fig.4g).

As a stringent test of the possibility that Wnt ligands stimulated expression of Flt1in deep RMCs,we flow-sorted the CD11b 1,F4/801,CD2042population from both control (Wls fl/1)and experimental (Wls fl/1;cfms–icre )mice at P12and performed quantitative PCR

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

Regulation of angiogenesis by a non-canonical Wnt–

for

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INL OPL Figure 4|Flt1expression in myeloid cells is regulated by a Wnt pathway.a ,PCR for Flt1transcript in flow-sorted RMCs.b -act ,b -actin transcript.b ,P14retinal sections labelled for Flt1and Iba1.Scale bars,25m m.c ,P18vessel branch points in the superficial and deep vasculature in Flt1fl/1and Flt1fl/1;cfms–icre mice (n 57,Student’s t -test).n 57.d ,Isolectin labelling of the retinal vasculature in labelled genotypes.Scale bars,50m m.e ,PCR for indicated

transcripts in E11.5whole embryo (con)and RAW264.7cells.f ,g ,quantitative PCR (f )for soluble Flt1and membrane-tethered Flt1in Wnt-treated

RAW264.7cells and ELISA (g )for soluble Flt1on medium from Wnt-treated RAW264.7cells (n 54,one-way ANOVA).h ,Quantitative PCR for soluble Flt1in flow-sorted deep RMCs from Wls fl/1and Wls fl/1;cfms–icre mice (n 54,Student’s t -test).Errors are s.e.m.;NS,not significant.

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4|N A T U R E |V O L 000|00M O N T H 2011

Flt1transcripts.We were never able to amplify the membrane-tethered Flt1transcript but showed that there was a90%reduction in transcript level for soluble Flt1(Fig.4h).This was consistent with the maximal phenotype observed in the heterozygous conditional Wls mutants. Although a heterozygous phenotype is not unusual,it is perhaps sur-prising that conditional homozygosity gave no further significant change.This kind of response might be explained by a low signalling sensitivity threshold for the Wnt–Flt1pathway.

The data we describe show that RMCs can modulate angiogenic responses directly by producing the VEGF inhibitory receptor Flt1. Unexpectedly,we also show that the production of Flt1depends on myeloid non-canonical Wnt ligands.In this setting,myeloid Wnt ligands might function in autocrine stimulation as has been documented for cultured macrophages27or might operate via a more complex path-way involving another cell type as an intermediate.The Wnt–Flt1res-ponse represents a new pathway for the regulation of VEGF-stimulated angiogenesis.The targeting of appropriate Fzd receptors or other Wnt–Flt1pathway components may offer new opportunities to modulate the production of Flt1and thus,the VEGF-stimulated angiogenic response. Because macrophage-related cells are ubiquitous and highly mobile,it is possible that the Wnt–Flt1pathway will be a general means to suppress VEGF locally and thus to restrain vascular responses.In future studies it will be interesting to determine,for example,if this pathway is active in the suppression of wound angiogenesis by macrophages13or is inacti-vated in macrophage-dependent tumour angiogenesis28. Additionally,our observations are consistent with a string of recent papers describing the role of myeloid cells beyond their well-documented functions in innate immunity7,8,17,29.These findings build on an idea articulated nearly a century ago30.It was suggested then that phagocytes originally evolved to regulate developmental processes,and that their immune functions were a later evolutionary adaptation.Macrophages were proposed to be the‘‘policemen’’of multi-cellular organisms,and that they could establish‘‘harmony from chaos’’.Here we show,in the setting of the retina,that myeloid cells do just that by fine-tuning vascular density and directing vascular traffic.

METHODS SUMMARY

We prepared and stained retinas as reported previously1.To isolate RMCs,we digested retinas,pre-enriched with CD11b beads,and sorted for surface makers with the FACSAria II.To obtain conditioned medium from MEFs,we performed transient transfections with Wnt5a,Thy1.1and cre plasmids,sorted Thy1.1-positive MEFS,and re-plated transfected cells.We performed calcium imaging on RAW264.7cells loaded in Ringer’s solution with5m M Fura-2AM and imaged at 510nm at1Hz after excitation at340nm and380nm.All animal experiments were performed in accordance with IACUC-approved guidelines and regulations.

Full Methods and any associated references are available in the online version of the paper at http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.html/nature.

Received20July2010;accepted30March2011.

Published online29May2011.

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Supplementary Information is linked to the online version of the paper at

http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.html/nature.

Acknowledgements We thank P.Speeg for technical assistance and A.P.McMahon for the Wnt11mice.This work was supported by the NIH(J.A.S.,M.W-K.,J.W.P.,J.D.M.,T.Y., B.O.W.,R.A.L.)by the HHMI(J.D.M.)and Cancer Research UK(H.G.).

Author Contributions R.A.L provided project leadership and wrote the manuscript with J.A.S.J.A.S.,I.L.,S.R.,H.G.,and R.A.L.designed the experiments.J.A.S,I.L.,S.R.,G.M., A.C.C.,A.R.B.,J.F.,and R.A.performed the experiments.S.R.,J.W.P.,T.Y.,N.F.and B.O.W developed critical reagents.Experimental supervision and helpful discussions were provided by M.W-K.,J.D.M.,S.R.,J.W.P.,and H.G.

Author Information Reprints and permissions information is available at

http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.html/reprints.The authors declare competing financial interests:details accompany the full-text HTML version of the paper at http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.html/nature. Readers are welcome to comment on the online version of this article at

http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.html/nature.Correspondence and requests for materials should be addressed to R.A.L.(http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.htmlng@http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.html).

LETTER RESEARCH

00M O N T H2011|V O L000|N A T U R E|5

METHODS

Quantification of retinal vasculature and isolation of RMCs.Retinas were prepared and imaged as reported1except that Alexa Fluor488isolectin GS-IB4 (Invitrogen)was used to label retinal vessels and RMCs.Other antibodies included anti-endomucin(Santa Cruz,V.7C7),anti-GFP(Abcam),anti-Iba1(Wako), anti-CD204(AbD Serotec),and anti-Flt1(R&D).For quantification,we used 2003magnification images located at the retinal periphery between artery and vein.For each genotype,at least three fields were analysed from at least four animals from a minimum of two litters.Control and experimental animals were littermates.This minimized the effect of strain background in producing variation in vascular density31–34.For flow sorting,retinas were digested and sorted as reported16except that0.5mg ml21DNase II(Sigma Aldrich)was used. Furthermore,myeloid cells were pre-enriched using CD11b beads(Miltenyi Biotech).Cells were then incubated with anti-CD16/32(clone24.G2)for30min and labelled with monoclonal antibodies to phycoerythrin-Cy7conjugated anti-CD11b(clone M1/70),allophycocyanin-Cy7or peridinin chlorophyll A protein-Cy5.5conjugated anti-F4/80(clone BM8),Alexa-647conjugated anti-CD204 (clone2F8),and7-aminoactinomycin D.Cells were sorted with a FACSAria II running DiVa software.

RNA isolation and RT–PCR.RNA was isolated using RNeasy(Qiagen). Quantitative PCR was performed with QuantiTect SYBR green(Qiagen). Primers are listed in Supplementary Table1.

In vitro analysis.MEFs were isolated as described35from Wls fllfl mice36and transfected with combinations of2.5m g Wnt5a,Thy1.1and cre plasmids using TransIT-2020(Mirus).MEFs were sorted using magnetic beads for Thy1.1, replated and supernatant was collected after24h.RAW264.7cells(ATCC)were grown in DMEM(10%FBS)and treated with recombinant Wnt3a(10ng ml21, R&D)or Wnt5a(500ng ml21,R&D).ELISA was performed using the sVEGFR1 Quantikine kit(R&D).For calcium imaging,RAW264.7cells were loaded in Riger’s solution with5m M Fura-2AM and imaged at510nm at1Hz after excita-tion at340nm and380nm.Data was acquired using EasyRatioPro.Ca21traces are 50-cell averages from two experiments.

Statistics.All statistical tests used are stated in the figure legends.In analysing quantitative PCR data,the P values refer to a comparison of the DD C t values. Animals.Breeding and genotyping of Wls fl(ref.36),cfms–icre(ref.18),Z/EG (ref.37),Wnt5a1/2(ref.19),Wnt111/2(ref.20)and Flt fl(ref.26)was performed as previously described.Lrp5fl will be described in detail in a forthcoming pub-lication.The allele is a conventional design where exon2of the Lrp5gene is flanked by LoxP sites.It has been confirmed that deletion between the LoxP sites produces a loss-of-function.All animal experimentation was carried out using protocols approved by the Institutional Animal Care and Use Committee.

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and pigment epithelium–derived factor in Brown Norway and Sprague Dawley rats contributing to different susceptibilities to retinal neovascularization.Diabetes 51,1218–1225(2002).

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vasculature.Invest.Ophthalmol.Vis.Sci.45,441–447(2004).

34.Chan,C.K.et al.Differential expression of pro-and antiangiogenic factors in mouse

strain-dependent hypoxia-induced retinal http://www.wendangku.net/doc/28d220ae7375a417876f8f6d.htmlb.Invest.85,

721–733(2005).

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excision.Genesis28,147–155(2000).

RESEARCH LETTER