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1 DOI: /nc2331 PCre;Ros26R 12 h induction 48 h induction Vegfr3 i EC c d ib4 24 h induction VEGFR3 e Fold chnge P < 0.05 Vegfr3 i EC Vegfr3 Figure S1 Cre ctivtion leds to genetic deletion of Vegfr3 specificlly in the endothelium in the PCre;R3 flox/flox mice. () Cre expression detected y Xgl stining (lue) in P5 retins of PCre;R3 flox/flox ;Ros26R pups, fter 12 hours () or 48 hours () of 4OHT dministrtion. (cd) Cre expression results in deletion of the floxed Vegfr3 llele. VEGFR3 (red) nd isolectin B4 stining (green) of P5 retins. Cre ws induced for 48 hours. (e) Vegfr3 mrn levels in Vegfr3 i EC nd littermte retins fter 48 hours of Cre induction. Expression of Gpdh ws used s the normliztion control. P < 0.05; n = 4 Vegfr3 i EC nd 3 pups. Sclers: 100 µm. 1

2 Vegfr3 iδec V V Brdu ib4 V V Figure S2 Incresed endothelil prolifertion in Vegfr3 i EC mice. BrdU (red) nd isolectin B4 (green) stining of retins. = rtery, V = vein. Scler: 100 µm. 2

3 SV Zone Projection Vegfr3 iδec c % Vessel/ totl re 45 P < Vegfr3 iδec Brnches (SVZ)/ mm 2 P < 0.01 Pil Zone d Vegfr3 iδec Numer of vessels (pil side)/ mm P > Vegfr3 iδec Figure S3 Blood vsculr hyperplsi ut not excess sprouting in the hindrins of E12.5 emryos with trgeted deletion of Vegfr3 in the endothelium (), Endomucin stining (green) of E12.5 Vegfr3 i EC nd littermte hindrins fter 48 hours of 4OHT dministrtion. Scler: 100 µm. (d) Quntittive nlysis of the hindrins shown in (). () Endomucin positive surfce re/ totl re, (c) numer of vessel rnching points in the suventriculr zone, (d) numer of vessel sprouts on the pil side. Error rs = S.E.M. P < 0.01, P < 0.001; n = 5 Vegfr3 i EC nd 6 emryos. 3

4 PECM1 VEGFR3 Merged dvegfb dvegf Vegfr3 iδ EC Vegfr3 iδ EC Figure S4 Induction of VEGFR3 in ngiogenic lood vessels in the dult mouse er following denovirl VEGF gene trnsfer. Er skin trnsduction with denovirl gene trnsfer vectors encoding VEGF induces VEGFR3 expression in the ngiogenic vessels in wild type mice ut not in Vegfr3 i EC mice. PECM1 (green) nd VEGFR3 (red) stining of the skin of mouse ers fter denovirl trnsduction with the indicted vectors. rrows indicte VEGFR3 positive lood vessels. Cre ws induced y sucutneous implnttion of slow relese tmoxifen pellets for 11 dys. Scler: 100 µm. 4

5 VEGF αvegfr3 IgG kd IP: VEGFR2 kd py/vegfr2: 15 min 30 min 60 min R2 WB: py R2 WB: R2 mrn fold chnge P > 0.05 αvegfr3 IgG c mrn fold chnge Vegfc +/ Hey1 Hey2 Nrrp Dll4 Vegfc Vegf Figure S5 VEGFR3 locking ntiodies do not influence VEGF/VEGFR2 interctions in HUVECs in vitro nd they do not lter Notch signling in the retin endothelium in vivo; evlution of Vegfc expression in Vegfc heterozygous retins. (), Cultured humn umilicl vein endothelil cells (HUVECs) stimulted with VEGF in the presence of VEGFR3 locking ntiodies or control IgG for the indicted times. VEGFR2 ws immunoprecipitted (IP) followed y immunolotting (IB) for phosphotyrosine (py) nd VEGFR2. Numers elow the lots indicte reltive nd intensities of py to VEGFR2, normlized to the IgG control nd of the sme time point. Uncropped imges of lots re shown in Supplementry Figure S9c. (), Fold chnges in Hey1, Hey2, Nrrp nd Dll4 mrn levels nlyzed y RT qpcr in the retins of NMRI pups fter tretment with ntiodies locking VEGFR3 or nonspecific rt IgG (50 mg/kg for 48 hours prior to scrifice). Expression of Gpdh ws used s normliztion control. P > 0.05; n = 4 nd 3 pups in ntivegfr3 nd control Ig groups, respectively. Error rs = S.E.M. (c) Vegfc +/ heterozygotes disply reduced levels of Vegfc mrn, ut no chnge in Vegf levels. Vegfc nd Vegf mrn levels in Vegfc +/ nd wild type littermte retins t P5. Expression of Gpdh ws used s the normliztion control. P < 0.001; n = 3 Vegfc +/ nd 4 pups. Error rs = S.E.M. 5

6 ib4 Vegfd+/ Vegfd/ Vegfd+/ %Vsculr /totl re Sprouts/ vsculr re (mm 2 ) Brnches/ vsculr re (mm 2 ) Vegfd+/+ Vegfd+/+ Vegfd+/ Vegfd/ Vegfd+/ Vegfd/ Vegfd+/ Vegfd/ Vegfd+/+ Figure S6 Loss of Vegfd is dispensle for norml ngiogenesis. (), Isolectin B4 stining of Vegfd /, Vegfd +/ nd littermte P5 mouse retins. (), Quntittive nlysis of the retins shown in (): % isolectin B4 positive/ totl vessel re, sprout numer nd numer of vessel rnching points per vsculr re. Scler: 100 µm. P > 0.05; n = 3 Vegfd /, 5 Vegfd +/ nd 4 pups. Error rs = S.E.M. 6

7 VEGFC F4/80 ib4 Figure S7 Mcrophges regulte vsculr vessel fusion nd rnch stility vi VEGFC. () Triple immunofluorescence stining of ib4 (white), mcrophges (F4/80, green) nd VEGFC (red) of wild type retins t P5. F4/80 positive mcrophges re distriuted evenly in the retin, ut they express VEGFC only t the sites of vsculr rnching (rrows in [], rrowheds in []). Note mcrophge ridging two sprouts expressing low levels of VEGFC (rrow in []). Sclers: 100 µm nd 50 µm for () nd (), respectively. 7

8 mrn fold chnge P < 0.05 VEGF 1h VEGF 2h VEGFC 1h VEGFC 2h Control DLL4 NOTCH1 NOTCH4 Figure S8 VEGFC induces DLL4 in hbecs. Fold chnges in DLL4, NOTCH1 nd NOTCH4 mrn levels nlyzed y RT qpcr, in nonconfluent hbecs stimulted with VEGF (100 ng ml 1 ) or VEGFC (100 ng ml 1 ) for 1 or 2 hours fter strvtion. GPDH ws used s normliztion control. P < 0.05; n = 3 pltes/group. Error rs = S.E.M. 8

9 kd Figure 2 2 kd 2c kd 2d kd Figure 3d c kd Supplementry Figure Tmmel, Zrkd et l. Figure S9 Figure S9 Full view of the films used to detect the Western lots. The oxed regions indicte the nds shown in the figures. 9

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