© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1465-1472 doi:10.1242/dev.104539

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ABSTRACTHere we show that dynamin 2 (Dnm2) is essential for angiogenesisin vitro and in vivo. In cultured endothelial cells lacking Dnm2,vascular endothelial growth factor (VEGF) signaling and receptorlevels are augmented whereas cell migration and morphogenesis areimpaired. Mechanistically, the loss of Dnm2 increases focal adhesionsize and the surface levels of multiple integrins and reduces theactivation state of β1 integrin. In vivo, the constitutive or inducible lossof Dnm2 in endothelium impairs branching morphogenesis andpromotes the accumulation of β1 integrin at sites of failed angiogenicsprouting. Collectively, our data show that Dnm2 uncouples VEGFsignaling from function and coordinates the endocytic turnover ofintegrins in a manner that is crucially important for angiogenesis invitro and in vivo.

KEY WORDS: Angiogenesis, Endocytosis, Endothelium, Mouse

INTRODUCTIONDynamin (Dnm) is a GTPase essential for membrane fission leadingto clathrin-mediated endocytosis (Ferguson and De Camilli, 2012).There are three isoforms of Dnm in mammalian cells, Dnm1, Dnm2and Dnm3, which are encoded by distinct genes that aredifferentially expressed in various tissues (Ferguson et al., 2009).Mice deficient in Dnms have been generated and the loss ofneuronally enriched isoforms, Dnm1 and Dnm3, results in impairedneurotransmission due to a defect in synaptic vesicle endocytosis(Ferguson et al., 2007; Raimondi et al., 2011). The loss of Dnm2leads to early embryonic lethality and the loss of Dnm1/2 inpodocytes regulates renal glomerular filtration (Soda et al., 2012).

Recent evidence largely based on pharmacological inhibition ofDnm function (Gourlaouen et al., 2013; Lanahan et al., 2010;Sawamiphak et al., 2010; Wang et al., 2006; Wang et al., 2010)suggests that clathrin-mediated endocytosis is necessary for vascularendothelial growth factor (VEGF) signaling in vitro andangiogenesis in vivo. The VEGF receptor 2 (Vegfr2; Kdr – MouseGenome Informatics) receptor complex interacts with the Ephreceptor ligand ephrin B2, and this complex is internalized via

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1Vascular Biology and Therapeutics Program, Yale University School of Medicine,New Haven, CT 06520, USA. 2Department of Pharmacology, Yale UniversitySchool of Medicine, New Haven, CT 06520, USA. 3Department of VeterinaryPharmacology, Graduate School of Agriculture and Life Sciences, The Universityof Tokyo, Tokyo 113-8657, Japan. 4Cardiovascular Biology Program, OklahomaMedical Research Foundation, Oklahoma City, OK 73104, USA. 5Cell Biology, YaleUniversity School of Medicine, New Haven, CT 06520, USA. 6Howard HughesMedical Institute, Yale University School of Medicine, New Haven, CT 06520,USA.*These authors contributed equally to this work

‡Author for correspondence (william.sessa@yale.edu)

Received 4 October 2013; Accepted 5 February 2014

clathrin to promote angiogenesis (Wang et al., 2011b). Moreover,the cytoplasmic domain of ephrin B2 interacts with a complexcontaining clathrin, the general clathrin adaptor complex (AP2),Dab2, Dnm2 and myosin VI (Nakayama et al., 2013). Theendothelial-specific loss of Dab2 attenuates Vegfr2 internalizationand angiogenesis, and loss of aPKC increases endocytosis andsignaling, implying that Vegfr2 internalization is essential forsignaling and angiogenesis. However, there are additional datasuggesting that the cargo-specific endocytic clathrin adaptormolecules epsin 1 and epsin 2 can terminate VEGF signaling as thegenetic loss of epsins impedes Vegfr2 endocytosis, increases VEGFsignaling and promotes abnormal pro-angiogenic functions (Pasulaet al., 2012). Indeed, the interaction of epsins with ubiquitylatedVegfr2 allows for its clathrin-mediated internalization anddestruction. Clearly, the role of endocytosis in the angiogenesisresponse is complex and is influenced by diverse routes ofendocytosis and multiple cargo-specific clathrin adaptor molecules.However, the role of the central GTPase in endocytosis, Dnm2, hasnot been genetically dissected in endothelium.

Here, we show that Dnm2, the major isoform of Dnm expressedin endothelial cells (ECs), is essential for developmentalangiogenesis. In cultured ECs depleted of Dnm2, VEGF receptorendocytosis is impaired and VEGF signaling is enhanced. Despiteaugmented VEGF signaling, VEGF-induced cell migration andtubular morphogenesis is impaired, implying that pathwaysdownstream of Vegfr2 signaling are regulated by Dnm2.Mechanistically, the loss of Dnm2 or clathrin increases focaladhesion size and impairs integrin internalization thereby reducingcell motility and morphogenesis. Moreover, the loss of Dnm2 inendothelium in vivo attenuates branching angiogenesis and promotesthe accumulation of β1 integrin at sites of blunted angiogenicsprouts, replicating the data in cultured ECs. Thus, Dnm-dependentendocytosis uncouples VEGF signaling from angiogenic pathwaysand is crucial for the endocytic turnover of integrins andorganization of EC during angiogenesis in vivo.

RESULTS AND DISCUSSIONTo examine the role of endocytosis during angiogenesis, weinvestigated the expression of all three Dnm proteins in mouse lungECs (MLECs) and found that Dnm2 is the major isoform expressed(supplementary material Fig. S1A). Indeed, reducing DNM2 orclathrin heavy chain (CHC; CLTC – Human Gene NomenclatureDatabase) levels by 80-90% via siRNA in human umbilical veinECs (HUVECs) significantly reduced the endocytosis offluorescently labeled human transferrin, a well-characterized cargomolecule dependent on Dnm and clathrin (Ferguson et al., 2009)(Fig. 1A). DNM2 knockdown increased the surface accumulation ofactin nucleating protein p34 subunit of the Arp2/3 complex and theclathrin adaptor adaptin 2, consistent with data in fibroblasts

Dynamin 2 regulation of integrin endocytosis, but not VEGFsignaling, is crucial for developmental angiogenesisMonica Y. Lee1,2,*, Athanasia Skoura1,2,*, Eon Joo Park1,2, Shira Landskroner-Eiger1,2, Levente Jozsef1,2, Amelia K. Luciano1,2, Takahisa Murata3, Satish Pasula4, Yunzhou Dong4, Mohamed Bouaouina2, David A. Calderwood1,2,5, Shawn M. Ferguson4, Pietro De Camilli5,6 and William C. Sessa1,2,‡

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(Fig. 1B). The loss of DNM2 had no effect on cell viability orapoptosis (supplementary material Fig. S1B). As DNM2-dependentendocytosis has been implicated in VEGF signaling, HUVECstreated with scrambled or DNM2 siRNA were stimulated withVEGF165 (50 ng/ml) and downstream pathways examined. VEGFsignaling was increased in HUVECs lacking DNM2, asdemonstrated by enhanced phosphorylation of VEGFR2 (KDR –

Human Gene Nomenclature Database), PLCγ, ERK and AKT(Fig. 1C) and by VEGF-induced changes in intracellular calcium(Fig. 1D). Moreover, the loss of DNM2 increased surface VEGFR2levels as quantified by fluorescence-activated cell sorting (FACS)(Fig. 1E). Interestingly, despite enhanced VEGF signaling, serum-and VEGF-driven chemotaxis (Fig. 1F), and cord formation(Fig. 1G) are impaired whereas F-actin rich stress fibers are

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Fig. 1. Loss of Dnm2 regulates VEGF signaling but reduces angiogenesis. (A) Loss of DNM2 or CHC (50 nM of each) reduces transferrin endocytosis inHUVECs. n=4 experiments. Inset shows knockdown efficiency of corresponding siRNAs. (B) Localization of Arp2/3 complex and AP2 (adaptin 2) in control andDnm2-deficient HUVECs. Scale bars: 7.5 μm. (C-E) Effect of VEGF stimulation (50 ng/ml) on signaling. (C) Representative blot and quantification (from fourindividual experiments) showing enhanced VEGF signaling via analysis of phospho-proteins. (D,E) Calcium (D) and surface Vegfr2 (E) levels in Dnm2-depleted HUVECs (from four individual experiments). (F,G) Basal, serum-driven and VEGF-driven HUVEC migration (F) and cord formation (G) was markedlyimpaired by Dnm2 siRNA. Data are from three independent experiments repeated in duplicates. SCR, scrambled siRNA. Error bars represent s.e.m. *P

increased (supplementary material Fig. S1C) in cells that lackDNM2.

The reduction in cell motility, impaired morphogenesis andincreased stress fibers triggered by the loss of DNM2 suggested thatDNM2 may be regulating integrin-dependent adhesive properties.Previous work in other cell types have shown that clathrin-mediatedendocytosis is essential for integrin recycling and focal adhesiondisassembly and that Dnm2 can interact with clathrin adaptors or

focal adhesion kinase (FAK; Ptk2 – Mouse Genome Informatics) toregulate focal adhesions (Arjonen et al., 2012; Chao et al., 2010;Chao and Kunz, 2009; Ezratty et al., 2009; Ezratty et al., 2005;Nishimura and Kaibuchi, 2007; Wang et al., 2011a). Therefore, wetested whether Dnm2 could regulate adhesion dynamics and integrinendocytosis in primary ECs. Immunofluorescence staining forpaxillin and FAK (Fig. 2A) showed increased size of focal adhesions(quantified in Fig. 2B) in cells depleted of Dnm2 or CHC

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Fig. 2. Dnm2 regulates focal adhesion size and integrin internalization. (A,B) Representative images of paxillin and FAK accumulation in DNM2-deficientcells (A) and quantification of these data (B; triplicate transfections of siRNA from an experiment, repeated three additional times). (C) Loss of DNM2 or CHCincreases surface, but not total, levels of β1 integrin as detected by surface biotinylation (as described in Materials and Methods). Blot shown is representativeof four experiments. (D,E) FACS analysis of surface αvβ5, αvβ3 and β1 integrins after DNM2 knockdown in HUVECs (D) and quantification of these data (E).n=3-4 experiments. (F) Levels of active β1 integrin (via binding FN 9-11) relative to total β1 levels (integrin activation index) in HUVECs treated with DNM2siRNA were reduced in the absence of DNM2. Data represent results from three to four independent experiments. (G) Adhesion to fibronectin is reduced inDNM2 siRNA-treated cells. O.D., optical density. Data are from four independent experiments. SCR, scrambled siRNA. Error bars represent s.e.m. *P

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(supplementary material Fig. S2). As seen in Fig. 2C, reducinglevels of Dnm2 or CHC (Cltc – Mouse Genome Informatics) viasiRNA-enhanced β1 integrin levels at the cell surface (surfacefraction) without altering the total levels of this integrin in wholecell lysates. This was confirmed by FACS in non-permeabilizedcells using β1-, αvβ3- or αvβ5 integrin selective antibodies(Fig. 2D,E). To examine if the loss of Dnm2 has a net effect onintegrin activation, FACS (see supplementary material Fig. S3 forprofiles and gating) was performed using a GST fusion proteincomposed of fibronectin type III repeats 9 through 11 (FN 9-11),which selectively binds to activated integrins (Bouaouina et al.,2012). As seen in Fig. 2F, the loss of Dnm2 reduced the ratio ofactive to total pools of β1 integrin, implying that despite an elevationof surface integrins by the loss of Dnm2, the net activation state wasreduced. Finally, we examined whether the loss of Dnm2 affectedintegrin-matrix interactions by quantification of EC interactions withfibronectin, a ligand for β1 and β3 integrins. As seen in Fig. 2G, theloss of Dnm2 reduced EC adhesion to fibronectin, consistent withincreased levels of inactive integrins and this effect was also seenusing electric cell-substrate impedance sensing (ECIS) resistancemeasurements (supplementary material Fig. S4).

To study the role of Dnm2 function in angiogenesis in vivo, weinactivated Dnm2 in ECs via Cre/loxP-mediated recombination offloxed Dnm2 using a Tie2 CRE mouse (supplementary materialFig. S5A,B). EC deletion of Dnm2 caused gross morphologicaldefects at embryonic day (E) 9.5 and no live embryos remained atE11.5 (Fig. 3A-D). Major blood vessels, such as dorsal aortae, wereformed in Dnm2flox/flox, Tie2Cre mice, suggesting that Dnm2 is notrequired for the initial stages of vasculogenesis. However,Dnm2flox/flox; Tie2Cre embryos showed pale, abnormally wrinkledyolk sacs with a small number of blood-filled vessels at E9. Vesselsin the head and trunk exhibited defective vascular patterning(Fig. 3C,D) and the yolk sac was strikingly abnormal (Fig. 3E,F).As early as E9.0, tail intersomitic vessels (ISVs) of Dnm2flox/flox;Tie2Cre embryos (identical in somite number and size to littermates)failed to elongate properly resulting in reduced vessel length(supplementary material Fig. S5C). These data indicate that Dnm2function in ECs is required for mouse embryonic vascularmorphogenesis and remodeling.

Next, we used inducible loss of function in ECs to selectivelyexcise the Dnm2 gene by breeding Cdh5-CreERT2 mice (Beneditoet al., 2009) with floxed Dnm2 mice (Ferguson et al., 2009).Downregulation of Dnm2 expression was achieved by oral gavageof tamoxifen (2 mg) into pregnant Dnm2flox/flox females at either E9.5or E10.5 (Benedito et al., 2009; Pitulescu et al., 2010). Two daysafter the initiation of gene inactivation in ECs (E11.5-E12.5),Dnm2iΔEC embryos were macroscopically indistinguishable fromlittermate controls; however, detailed examination revealed theformation of subcutaneous hemorrhages (Fig. 3I). Histological serialcross-sections of E12.5 embryos revealed the presence of dilatedvessels filled with blood that diffused into the abluminal space(Fig. 3J, top right panel). Finally, deletion of Dnm2 at E12.5 led tosignificant impairments in branching in the hindlimb (Fig. 3J,middle panels) and yolk sac (Fig. 3J, bottom panels). Theseobservations demonstrate that the absence of Dnm2 impairsdevelopmental angiogenesis and vascular patterning in mouseembryo.

Postnatal inactivation [initiated at postnatal day (P) 1 andanalyzed at P5] of Dnm2 in Dnm2iΔEC mice (P5) displayed impairedretinal vascular development demonstrated by reduced endothelialcell coverage, delayed radial growth towards the periphery(Fig. 4A,D), reduced branching (Fig. 4E,F) and impaired

filopodia/tip cells at the sprouting angiogenic front of the retina, butno significant differences in the thickness of the vessels andproliferation (supplementary material Fig. S6A,B) based onphospho-histone-3 staining (Fig. 4C). Dnm2iΔEC pups (P5) were asimilar weight to littermate controls (supplementary materialFig. S6C). The loss of Dnm2 had no effect on the distribution ofNG2 (Cspg4 – Mouse Genome Informatics)-positive pericytes andGFAP-positive astrocytes (supplementary material Fig. S6D,E).Furthermore, Dnm2 inactivation (P5-P9) showed that remodeling ofestablished vessels in the superficial capillary plexus at P12 isstrongly altered (Fig. 4G) and this difference was not due to growthimpairment (supplementary material Fig. S6F). Centrifugaloutgrowth and sprouting of the nerve fiber layer (NFL) vasculatureinto the outer plexiform layer (OPL) was compromised in Dnm2iΔEC

retinas resulting in cyst-like outgrowths (Fig. 4H, asterisks).Collectively, these data demonstrate that the postnatal lack of Dnm2in endothelium diminishes sprouting angiogenesis.

Finally, to examine whether Dnm2 regulates the localization anddistribution of Vegfr2 and β1 integrin in vivo, Dnm2 was inactivatedby injection of tamoxifen at P1-P3 and FACS was carried out forsurface β1 integrins and Vegfr2 levels in retinal single cell isolatesat P6. The loss of Dnm2 increased the surface expression of bothproteins in vivo as seen in Fig. 4I,J. Moreover, β1 integrin wasdiffusely distributed throughout the growing vascular networks andat this time point did not show preference for expression in cells atthe growing front versus mature stalk cells (Fig. 4K). However, inretinal ECs lacking Dnm2, there was a marked accumulation of β1integrin in blunted tip cells near the angiogenic front. Thus, Dnm2-dependent endocytosis is crucial for the proportional turnover ofVegfr2 and β1 integrin needed for sprouting angiogenesis in vivo.

Collectively, the data reveal a major role for Dnm2 in endothelialcell functions in vitro and developmental angiogenesis in vivo. Ourgenetic data in conjunction with results from endothelial-specificepsin-deficient mice (Pasula et al., 2012) indicates that endocytosisnegatively regulates VEGF signaling. Epsins selectively bind toubiquitylated Vegfr2, but not integrins, permitting increased Vegfr2levels, signaling and function, whereas the loss of Dnm2 blocksendocytosis of both Vegfr2 and integrins, resulting in reducedangiogenesis. These data imply that a dominant function of Dnm2during angiogenesis is to regulate the assembly/disassembly of cell-matrix adhesions, key molecular events that promote endothelialmorphogenesis and vessel stability.

Upon first glance, our data may appear to contradict previouswork showing that Vegfr2 endocytosis is necessary for signaling andangiogenesis. Previously, Lanahan et al. (Lanahan et al., 2010)documented that the loss of synectin or myosin VI did not affect themovement of Vegfr2 from the cell surface to the endosome butindeed reduced, not eliminated, intracellular VEGF signaling.Lampugnani et al. (Lampugnani et al., 2006) showed that the lossof clathrin reduced Vegfr2 endocytosis, but VEGF signaling wasonly suppressed in cells lacking VE-cadherin (also known ascadherin 5), not in normal ECs. In Wang et al. (Wang et al., 2010),the authors injected dynasore, a Dnm GTPase inhibitor, into thedeveloping retina to show that Dnm was required for Vegfr2signaling; however, a recent paper now shows this class of inhibitorsblock endocytosis in cells lacking all Dnms, showing that theseinhibitors are non-specific (Park et al., 2013). Our work and thework on epsins clarifies that blocking endocytosis using discretemolecular tools increases VEGF signaling, consistent with a recentpaper showing that Dnm2 negatively regulates epidermal growthfactor receptor (EGFR) signaling (Sousa et al., 2012). However,once signaling is initiated at the cell surface, the subsequent

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endocytosis of Vegfr2 can promote additional post-plasmamembrane signaling.

Mechanistically, impairments in integrin internalization andinactivation are likely to explain the defective morphogenesis andremodeling observed in Dnm2iΔEC mice. Indeed, endothelial celldeletion of the β1 integrin subunit leads to early lethality (E9.5)with defects in vascular branching phenocopying the Dnm2flox/flox;Tie2Cre vascular defects (Carlson et al., 2008; Lei et al., 2008;Tanjore et al., 2008; Zovain et al., 2010). In the present paper, theloss of Dnm2 impedes endocytosis of the three integrins tested;thus, the loss of Dnm2 retards the endocytosis and recycling of

integrins, resulting in the net accumulation of inactive integrins.However, Dnm2 regulation of integrins is not likely to be anexclusive mechanism because endocytosis of ephrin B2, Notch andNotch ligands or VE-cadherin may also contribute to the Dnm2knockout phenotypes (Alghisi et al., 2009; Wang et al., 2006;Wang et al., 2011b). In summary, our results provide the firstdefinitive molecular insights into the role of Dnm-mediatedendocytosis in angiogenesis in vivo and show that VEGF signalingcan be uncoupled from pro-angiogenic endothelial cell functions,suggesting that endocytosis may be an ‘Achilles heel’ to attackpathological angiogenesis in disease.

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Fig. 3. Dnm2 function in ECs is required for vascular morphogenesis. (A,B) Deletion of Dnm2 in ECs caused severe embryo growth retardation at E9.5.(C,D) Dnm2flox/flox; Tie2Cre embryos (E9.5) showed defective vascular patterning. (E,F) Dnm2flox/flox; Tie2Cre yolk sacs exhibited abnormal vascular structures(E9.5). (G,H) At E9.0, tail intersomitic vessels (ISVs) of Dnm2flox/flox; Tie2Cre embryos showed significantly reduced vessel length. (I) Dnm2iΔEC embryosdevelop subcutaneous hemorrhages at E9.5-E11.5 and at E10.5-E12.5. (J) Histological serial cross-sections of E12.5 after tamoxifen (TMX) administration atE10.5. Dnm2iΔEC embryos showed dilated vessels and blood diffusion (top panel) and an impairment in vessel morphogenesis in the hindlimb (middle panel)and yolk sac vascular beds (bottom panel E12.5). A, aorta, V, cardinal Vein. Scale bars: 2 mm (A,B,I); 240 μm (C,D); 100 μm (E-H); 50 μm (J).

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MATERIALS AND METHODSRNAi experiments and immunofluorescenceHUVECs (P2-P6) were transfected by Oligofectamine (Invitrogen)according to the manufacturer’s instructions with siRNA of control(luciferase), human DNM2 or clathrin heavy chain (CHC) designed byQiagen. For endocytosis experiments, human Transferrin Alexa 568

(Invitrogen, T13343) was added to cells and incubated at 4°C for 15minutes (5 mg/ml stock, 1:400). Plates were transferred back to 37°C andincubated for 30 minutes. To remove cell surface-bound transferrin, cellswere washed with cold PBS, pH 2.7, containing 25 mM glycine and 3%bovine serum albumin and fixed with 4% paraformaldehyde for 15minutes, on ice.

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Fig. 4. Dnm2 is crucial for retinal angiogenesis and β1 integrin levels in vivo. (A) Representative images of reduced radial retinal EC growth in Dnm2iΔECpups (P5). (B) Emerging tip cell front of angiogenic vessels (P5). (C) Lack of Dnm2 in EC does not influence growth, as determined by PH3 staining.(D) Quantification of data shown in A. (B,E,F) Loss of Dnm2 in ECs leads to reduced branching (E,F; control, n=4 and Dnm2iΔEC, n=11) and impaired filopodia.(G,H) Remodeling of established vessels in the superficial capillary plexus at P12 is strongly altered in Dnm2iΔEC pups (100 μg TMX, P5-P12). P12 Dnm2iΔECpups with compromised centrifugal outgrowth (H). Centrifugal outgrowth of the nerve fiber layer (NFL) and compromised sprouting into the outer plexiform layer(OPL). Zooming into the NFL shows cyst-like outgrowths in Dnm2iΔEC pups. (I,J) FACS plots of retinal cells isolated from P6 mice showing elevated surfaceVegfr2 (I) and β1 integrin (J) in Dnm2iΔEC pups. (K) Distribution of β1 integrin is abnormal in Dnm2iΔEC retinas. *P

Biotin surface labelingBiotin cell surface labeling was performed on HUVECs 72 hours post-siDnm2 transfection. In brief, cells were incubated with a 1 mM EZ-link-sulfo-NHS-S-S-biotin solution (Pierce #21331) for 30 minutes on ice. Cellswere washed with a 50 mM glycine/PBS solution, pH 7.2 and subsequentlyharvested in protein lysis buffer. Following centrifugation, an aliquot wascollected for protein levels in total cell lysates, and the biotin-labeled surfaceproteins were incubated with Neutravidin Protein agarose beads (Pierce#29200) at 4°C overnight. Flow-through fractions were collected after theincubation period following a 3000 rpm (960 g) spin. The beads were thenwashed several times in lysis buffer prior to addition of Laemmli Buffer forimmunoblot analysis of β1 integrin (BD Biosciences).

Analysis of integrin activation and expressionsiRNA-treated HUVECs were used for analysis of surface expression andintegrin activation by measuring the binding of a fibronectin fragment (FN9-11) as described previously (Bouaouina et al., 2012). Total β1, αVβ3 andαVβ5 integrin expression levels were measured in parallel by staining,respectively, with anti-β1 (clone TS2/16, Biolegend), anti-αVβ3 (clone23C6, Biolegend) and anti-αVβ5 (clone 15F11, Millipore) antibodies. BoundFN9-11 and integrin expression were detected with Allophycocyanin (APC)-conjugated streptavidin (Thermoscientific) or AlexaFluor647-conjugateddonkey anti-mouse IgG, respectively, as described in supplementary materialFig. S3.

Generation of Dnm2 conditional deletion in the endotheliumDnm2 conditional knockout targeting strategy was previously reported(Ferguson et al., 2009). EC Dnm2-deficient mice were generated by crossingDnm2flox/flox females with Dnm2flox/+; Tie2Cre males (Jackson Laboratory).Tamoxifen (Sigma, T5648)-inducible Dnm2iΔEC were obtained by breedingDnm2flox/flox females with Dnm2flox/flox;Cdh5-CreERT2 male mice. Pregnantfemales were treated with TMX by oral gavage (2 mg, Sigma T5648) andembryos (E11.5-E12.5) were fixed in 4% PFA/PBS, embedded in OCT andcut into 8 µm serial cross-sections. All experimental procedures wereapproved by the Yale University Institutional Animal Care Use Committee.

Immunohistochemistry and immunofluorescence of mousetissuesHematoxylin and Eosin staining was performed with Mayer’s Hematoxylin(10 minutes) and counterstained with Eosin (1 minute). Images wereprocessed with a Nikon Eclipse 80i microscope and using the Photoshop CSsoftware. For whole-mount staining, freshly isolated embryos and yolk sacswere fixed for 10 minutes in 4% PFA/PBS at 4°C, washed in PBS andincubated in blocking/permeabilization solution [PBS (pH 7.2), 1% BSA,0.1% Triton X-100] for 1 hour. Embryos were incubated overnight at 4°Cin the presence of PE-conjugated rat anti-mouse CD31 (BD Pharmingen553373). Microscopy was carried out with a confocal laser scanning LeicaTCS SP5 microscope. ISV length quantification was performed using theImage J software (National Institutes of Health, NIH).

Adhesion assayNinety-six-well plates were coated with fibronectin (5 μg/ml, 354008,Becton Dickinson) at 4°C overnight. Plates were blocked with 10 mg/mlheat-denatured bovine serum albumin for 30 minutes. A set volume (100 μl)of HUVECs in cell suspension (5×105 cells/ml in basal medium with 0.1%BSA) were added onto plates and incubated for 30 minutes at 37°C. Afterincubation, non-adherent cells were removed by rinsing with PBS. Attachedcells were fixed in 4% PFA/PBS for 20 minutes and stained with 0.1%Crystal Violet. Crystal Violet was dissolved in 10% acetic acid, and plateswere read at an absorbance reading of 575 nm.

For Electrical Cell Impedance System (ECIS, Applied Biophysics)measurements, HUVECs in suspension treated with either SCR or siDnm2were plated in suspension onto polycarbonate wells containing gold electrodes(1×105 cells/well, in normal growth medium). Endothelial resistancemeasurements as a function of time were recorded as described bymanufacturers. In brief, a 4000 Hz current was applied across the electrodeswhere the in-phase and out-of-phase voltages were monitored in real-time andsubsequently converted to measurements of trans-endothelial resistance.

Mouse retina vascular system analysisFor retina staining, procedures were followed as described (Pitulescu etal., 2010) with minor modifications. Pups (P1-P3) were injected with 50μg tamoxifen and for later stages (P12) a 100 μg was used (P5-P9).Briefly, retinas were dissected out, fixed, permeabilized and stained withAlexa-594-conjugated isolectin B4 (Invitrogen, I21413; 1:200), rabbitanti-phospho-histone H3 (Cell Signaling, 9701; 1:200), rabbit anti-GFAP(Neomarkers, RB-087-A0; 1:200) and rabbit anti-NG2 (Millipore,AB5320; 1:200).

Whole retina FACSRetinas of control or Dnm2iEC mice were excised at P6 (n=5-7 per group)and digested in Type I Collagenase for 45 minutes at 37°C (Sigma), filteredvia a 40-μm cell strainer and washed with PBS/0.5% fetal bovine serum, pH7.4. Cells were blocked with mouse BD Fc Block (BD Pharmingen,553142), followed by staining for either phycoerythrin (PE)-conjugatedVEGFR-2 (BD Pharmingen) or PE-conjugated Integrin β1 (eBiosciences).Cells were measured by FACSCalibur flow cytometer and analyzed usingFlowJo software.

Statistical analysesStatistical analyses were performed with Prism 5 software (GraphPad) usingthe two-tailed, unpaired Student’s t-test or one-way ANOVA, whenappropriate. P values

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Ferguson, S. M., Brasnjo, G., Hayashi, M., Wölfel, M., Collesi, C., Giovedi, S.,Raimondi, A., Gong, L. W., Ariel, P., Paradise, S. et al. (2007). A selective activity-dependent requirement for dynamin 1 in synaptic vesicle endocytosis. Science 316,570-574.

Ferguson, S. M., Raimondi, A., Paradise, S., Shen, H., Mesaki, K., Ferguson, A.,Destaing, O., Ko, G., Takasaki, J., Cremona, O. et al. (2009). Coordinated actionsof actin and BAR proteins upstream of dynamin at endocytic clathrin-coated pits.Dev. Cell 17, 811-822.

Gourlaouen, M., Welti, J. C., Vasudev, N. S. and Reynolds, A. R. (2013). Essentialrole for endocytosis in the growth factor-stimulated activation of ERK1/2 inendothelial cells. J. Biol. Chem. 288, 7467-7480.

Lampugnani, M. G., Orsenigo, F., Gagliani, M. C., Tacchetti, C. and Dejana, E.(2006). Vascular endothelial cadherin controls VEGFR-2 internalization and signalingfrom intracellular compartments. J. Cell Biol. 174, 593-604.

Lanahan, A. A., Hermans, K., Claes, F., Kerley-Hamilton, J. S., Zhuang, Z. W.,Giordano, F. J., Carmeliet, P. and Simons, M. (2010). VEGF receptor 2 endocytictrafficking regulates arterial morphogenesis. Dev. Cell 18, 713-724.

Lei, L., Liu, D., Huang, Y., Jovin, I., Shai, S.-Y., Kyriakides, T., Ross, R. S. andGiordano, F. J. (2008). Endothelial expression of beta1 integrin is required forembryonic vascular patterning and postnatal vascular remodeling. Mol. Cell. Biol. 28,794-802.

Nakayama, M., Nakayama, A., van Lessen, M., Yamamoto, H., Hoffmann, S.,Drexler, H. C., Itoh, N., Hirose, T., Breier, G., Vestweber, D. et al. (2013). Spatialregulation of VEGF receptor endocytosis in angiogenesis. Nat. Cell Biol. 15, 249-260.

Nishimura, T. and Kaibuchi, K. (2007). Numb controls integrin endocytosis fordirectional cell migration with aPKC and PAR-3. Dev. Cell 13, 15-28.

Park, R. J., Shen, H., Liu, L., Liu, X., Ferguson, S. M. and De Camilli, P. (2013).Dynamin triple knockout cells reveal off target effects of commonly used dynamininhibitors. J. Cell Sci. 126, 5305-5312.

Pasula, S., Cai, X., Dong, Y., Messa, M., McManus, J., Chang, B., Liu, X., Zhu, H.,Mansat, R. S., Yoon, S. J. et al. (2012). Endothelial epsin deficiency decreasestumor growth by enhancing VEGF signaling. J. Clin. Invest. 122, 4424-4438.

Pitulescu, M. E., Schmidt, I., Benedito, R. and Adams, R. H. (2010). Inducible genetargeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.Nat. Protoc. 5, 1518-1534.

Raimondi, A., Ferguson, S. M., Lou, X., Armbruster, M., Paradise, S., Giovedi, S.,Messa, M., Kono, N., Takasaki, J., Cappello, V. et al. (2011). Overlapping role ofdynamin isoforms in synaptic vesicle endocytosis. Neuron 70, 1100-1114.

Sawamiphak, S., Seidel, S., Essmann, C. L., Wilkinson, G. A., Pitulescu, M. E.,Acker, T. and Acker-Palmer, A. (2010). Ephrin-B2 regulates VEGFR2 function indevelopmental and tumour angiogenesis. Nature 465, 487-491.

Soda, K., Balkin, D. M., Ferguson, S. M., Paradise, S., Milosevic, I., Giovedi, S.,Volpicelli-Daley, L., Tian, X., Wu, Y., Ma, H. et al. (2012). Role of dynamin,synaptojanin, and endophilin in podocyte foot processes. J. Clin. Invest. 122, 4401-4411.

Sousa, L. P., Lax, I., Shen, H., Ferguson, S. M., De Camilli, P. and Schlessinger, J.(2012). Suppression of EGFR endocytosis by dynamin depletion reveals that EGFRsignaling occurs primarily at the plasma membrane. Proc. Natl. Acad. Sci. USA 109,4419-4424.

Tanjore, H., Zeisberg, E. M., Gerami-Naini, B. and Kalluri, R. (2008). β1 integrinexpression on endothelial cells is required for angiogenesis but not forvasculogenesis. Dev. Dyn. 237, 75-82.

Wang, Y., Jin, G., Miao, H., Li, J. Y., Usami, S. and Chien, S. (2006). Integrinsregulate VE-cadherin and catenins: dependence of this regulation on Src, but not onRas. Proc. Natl. Acad. Sci. USA 103, 1774-1779.

Wang, Y., Nakayama, M., Pitulescu, M. E., Schmidt, T. S., Bochenek, M. L.,Sakakibara, A., Adams, S., Davy, A., Deutsch, U., Lüthi, U. et al. (2010). Ephrin-B2controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483-486.

Wang, Y., Cao, H., Chen, J. and McNiven, M. A. (2011). A direct interaction betweenthe large GTPase dynamin-2 and FAK regulates focal adhesion dynamics inresponse to active Src. Mol. Biol. Cell 22, 1529-1538.

Zovein, A. C., Luque, A., Turlo, K. A., Hofmann, J. J., Yee, K. M., Becker, M. S.,Fassler, R., Mellman, I., Lane, T. F. and Iruela-Arispe, M. L. (2010). Beta1 integrinestablishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism. Dev. Cell 18, 39-51.

RESEARCH REPORT Development (2014) doi:10.1242/dev.104539

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Fig.€1. LossFig.€2. Dnm2Fig.€3. Dnm2Fig.€4. Dnm2Biotin surface labelingAnalysis of integrin activation and expressionGeneration of Dnm2 conditional deletion in the endotheliumImmunohistochemistry and immunofluorescence of mouse tissuesAdhesion assayMouse retina vascular system analysisWhole retina FACSStatistical analyses