© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1417 doi:10.1242/dev.108969

CORRECTION

Abnormal embryonic lymphatic vessel development in Tie1hypomorphic miceXianghu Qu, Kevin Tompkins, Lorene E. Batts, Mira Puri and H. Scott Baldwin

There was an error published in Development 137, 1285-1295.

Author name H. Scott Baldwin was incomplete. The correct author list appears above.

The authors apologise to readers for this mistake.

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INTRODUCTIONThe lymphatic vascular system is a blind-ended network ofendothelial cell-lined vessels essential for the maintenance of tissuefluid balance, immune surveillance and absorption of fatty acids inthe gut. The lymphatic vessels are also involved in the pathogenesisof diseases such as tumor metastasis, lymphedema and variousinflammatory conditions. Despite central roles in both normal anddisease physiology, our understanding of the development andmolecular regulation of the lymphatic vasculature lags far behindthat of the parallel blood vascular system (Oliver, 2004; Oliver andAlitalo, 2005).

Many details about the development of the lymphatic vasculaturehave been described only within the past decade, largely as a resultof studies of gene-targeted mice (Oliver and Srinivasan, 2008). Formammals, the development of the lymphatic vessels in embryos isinitiated when a subset of endothelial cells in the cardinal vein sproutto form the primary lymph sacs (Oliver and Detmar, 2002),confirming speculation of a venous origin made over a century ago(Sabin, 1909). In mice, the initiation of lymphatic differentiationis first discernible at embryonic day 10.5 (E10.5), when asubpopulation of endothelial cells (ECs) expressing Lyve1, Prox1(Wigle and Oliver, 1999), Sox 18 (Francois et al., 2008; Hosking etal., 2009) and Vegfr3 are detected on one side of the anterior cardinal

vein. These differentiating lymphatic endothelial cells (LECs)sprout, migrate and proliferate to form primary lymph sacs in thejugular region (Oliver, 2004). Subsequently, several lymph sacs areformed close to major veins in different regions of the embryo. Theprimary lymphatic vascular plexus undergoes remodeling andmaturation to create a mature lymphatic network composed of largelymph vessels as well as an extensive lymphatic capillary network.Numerous signaling proteins have been identified as important inthese later stages of the lymphatic development including ephrin B2(Makinen et al., 2005), neuropilin 2 (Yuan et al., 2002), angiopoietin2 (Gale et al., 2002; Dellinger et al., 2008), podoplanin (Schacht etal., 2003), integrin alpha 9 (Huang et al., 2000), and the transcriptionfactors Foxc2 (Petrova et al., 2004), Net (Ayadi et al., 2001), Vezf1(Kuhnert et al., 2005), adrenomedullin (Fritz-Six et al., 2008) andAspp1 (Hirashima et al., 2008).

The orphan receptor tyrosine kinase (RTK) Tie1 shares a highdegree of homology and is able to form heterodimers with Tie2, thereceptor for the angiopoietins (Yancopoulos et al., 2000; Peters etal., 2004), and is known to play a major role in vasculardevelopment. Genetic studies in mice have demonstrated that Tie1is required for development and maintenance of the vascular systemas mice lacking Tie1 die in mid-gestation of hemorrhage anddefective microvessel integrity (Puri et al., 1995; Sato et al., 1995).Expression of Tie1 is restricted to ECs and to some hematopoieticcell lineages (Partanen et al., 1992; Korhonen et al., 1994; Dumontet al., 1995; Hashiyama et al., 1996; Taichman et al., 2003; Yano etal., 1997). Interestingly, there is evidence that Tie1 is also expressedby the initial and collecting lymphatic vessels in adult mice (Iljin etal., 2002) and the first visible defect in the Tie1 mutants is edema(Sato et al., 1995). These observations led us to hypothesize thatTie1 might serve a unique function in the development ormaintenance of the lymphatic vasculature.

Development 137, 1285-1295 (2010) doi:10.1242/dev.043380© 2010. Published by The Company of Biologists Ltd

1Division of Pediatric Cardiology, Vanderbilt University Medical Center, Nashville,TN 37232, USA. 2Sunnybrook and Women’s College Health Sciences Center,University of Toronto, Toronto, Ontario M4N 3M5, Canada. 3Department of Cell andDevelopmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232,USA.

*Author for correspondence (scott.baldwin@vanderbilt.edu)

Accepted 5 February 2010

SUMMARYTie1 is an endothelial receptor tyrosine kinase that is essential for development and maintenance of the vascular system; however,the role of Tie1 in development of the lymphatic vasculature is unknown. To address this question, we first documented that Tie1 isexpressed at the earliest stages of lymphangiogenesis in Prox1-positive venous lymphatic endothelial cell (LEC) progenitors. LEC Tie1expression is maintained throughout embryonic development and persists in postnatal mice. We then generated two lines of Tie1mutant mice: a hypomorphic allele, which has reduced expression of Tie1, and a conditional allele. Reduction of Tie1 levels resultedin abnormal lymphatic patterning and in dilated and disorganized lymphatic vessels in all tissues examined and in impairedlymphatic drainage in embryonic skin. Homozygous hypomorphic mice also exhibited abnormally dilated jugular lymphatic vesselsdue to increased production of Prox1-positive LECs during initial lymphangiogenesis, indicating that Tie1 is required for the earlystages of normal lymphangiogenesis. During later stages of lymphatic development, we observed an increase in LEC apoptosis inthe hypomorphic embryos after mid-gestation that was associated with abnormal regression of the lymphatic vasculature.Therefore, Tie1 is required for early LEC proliferation and subsequent survival of developing LECs. The severity of the phenotypesobserved correlated with the expression levels of Tie1, confirming a dosage dependence for Tie1 in LEC integrity and survival. Nodefects were observed in the arterial or venous vasculature. These results suggest that the developing lymphatic vasculature isparticularly sensitive to alterations in Tie1 expression.

KEY WORDS: Tie1, Lymphatic development, Hypomorphic, Proliferation, Apoptosis, Mouse

Abnormal embryonic lymphatic vessel development in Tie1hypomorphic miceXianghu Qu1, Kevin Tompkins1, Lorene E. Batts1, Mira Puri2 and Scott Baldwin1,3,*

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In this study, we found that Tie1 was expressed in the Prox1-positive venous LEC progenitors and lymphatic vesselsthroughout embryonic and postnatal life. To circumvent earlyembryonic lethality observed in homozygous mutant animals, wegenerated mice with a conditional Tie1 allele that fortuitouslyresulted in hypomorphic expression of Tie1. We were able to takeadvantage of this Tie1 hypomorphic allele to demonstrate a uniquerole for Tie1 in lymphatic development that was not observed inthe arterial or venous vasculature. Furthermore, the severity of thephenotypes observed correlated with the expression level of Tie1.Our studies show that Tie1 is required for the early stages ofnormal lymphangiogenesis and is also involved in the laterremodeling and stabilization of lymphatic vessels in a dosage-dependent manner.

MATERIALS AND METHODSGeneration of Tie1 mutant allelesTo generate Tie1loxP/loxP mice, a Tie1 floxed targeting vector was constructedbased on the 129-Sv mouse genomic fragment used by Puri et al. (Puri et al.,1995). A 940 bp HpaI-SacI fragment containing a Tie1 minimal promoter(Korhonen et al., 1995; Iljin et al., 2002) and exon 1 (containing the initialATG codon) was inserted into the floxed KpnI-ClaI sites of the pDELBOYplasmid (pDELBOY-3X), which contains an Frt-site-flanked neomycingene (see Fig. S1 in the supplementary material). The targeting vector waselectroporated into 129 R1 embryonic stem (ES) cells (Nagy et al., 1993)and targeting was confirmed by Southern blotting with both 5� and 3�external probes and PCR with the primers 5�-ATGCCTGTTCT AT -TTATTTTTCCAG-3� and 5�-TCGGGCGCGTTCAGAGTGGTAT-3�.Correctly targeted cells were then injected into C57/BL6 blastocysts and twoseparate clones were found to transmit the targeted allele through thegermline. Both lines were maintained on a 129-Sv and C57/BL6 backgroundand demonstrated identical phenotypes.

Wholemount immunohistochemistryHearts, diaphragm, limbs and head skin were collected from embryos andprocessed as previously described (Gale et al., 2002). The following primaryantibodies were used: rat anti-mouse Pecam1 (Pharmingen, monoclonalMEC13.3), goat anti-mouse Vegfr3 antiserum (R&D Systems, #AF743),rabbit anti-Lyve1 (Upstate, #07-538) and Cy3-conjugated anti-a-smoothmuscle actin (SMA; Sigma, C-6198).

For fluorescence immunostaining, the following secondary antibodieswere used: Alexa Fluor 488-conjugated goat anti-rabbit (Molecular Probes,A-11008), Cy3-conjugated donkey anti-goat and Cy2-conjugated donkeyanti-rat IgG (Jackson ImmunoResearch Laboratories, #705-165-147 and#712-225-150). Tissues were mounted in Vectashield (Vector Laboratories)and analyzed using a fluorescent microscope (Olympus) while images werecaptured using a Zeiss LSM 510 confocal microscope.

To visualize anti-Lyve1 staining with light microscopy, biotinylated goatanti-rabbit IgG (Vector Laboratories, BA-1000) and biotinylated rabbit anti-goat IgG (Vector Laboratories, BA-5000) secondary antibodies were usedin horseradish peroxidase stainings with the Vectastain Kit (Vector Elite PK-6100) and DAB Kit (Vector Laboratories, SK-4100).

X-gal staining, BrdU incorporation, TUNEL staining and sectionimmunohistochemistryX-gal staining was performed on frozen sections of tissues fixed in 4%PFA/PBS from Tie1+/lacZ embryos as previously described (Puri et al., 1995)and tissues were then immunostained for lymphatics with primary antibodygoat anti-mouse Vegfr3 and fluorescently labeled secondary antibody Cy3-conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories,#705-165-147).

BrdU incorporation was assessed in embryos following intraperitonealinjection of pregnant females with BrdU. For immunohistochemistry, mouseanti-BrdU monoclonal antibody (clone G3G4, Developmental StudiesHybridoma Bank, University of Iowa, USA) and Alexa Fluor Cy2-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, #715-225-151) were used. Simultaneously, slides were also incubated with goat anti-

mouse Vegfr3 antibody or rabbit anti-Prox1 and Alexa Fluor 594-conjugateddonkey anti-goat IgG (Molecular Probes, A-11058) or Alexa Fluor 555 goatanti-rabbit IgG (Molecular Probes, A-21428). Lymphatic endothelial cell(EC) proliferation was also determined by labeling with Prox1 and Ki67(BD Biosciences, 550609). Other primary antibodies used in tissue sectionimmunohistochemistry were rat anti-mouse CD34 (eBioscience, 14-0341),rat anti-mouse Icam1 (eBioscience, 14-0542), rat anti-mouse endoglin(eBioscience, 14-1051) and rat anti-mouse VE-cadherin (BD Biosciences,555289). The percentage of proliferative lymphatic ECs in the cardinal veinand jugular lymph sac areas of embryos at E11.5 to E13.5 was defined as thenumber of BrdU+/Prox1+ cells divided by the total number of Prox1+ cellsin each field at similar level.

Apoptosis in lymphatic ECs was assed by TUNEL assay using ApopTagPlus Fluorescein in situ Cell Death Detection Kit (CHEMICONInternational, S7111). In addition, cleaved caspase 3 (Cell SignalingTechnology, 9661) and Vegfr3 double staining was used to detect apoptoticLECs on frozen sections. Images were acquired on an Olympus fluorescentmicroscope and processed in Adobe Photoshop. Percent apoptotic cells wasdetermined by blinded quantification of TUNEL-positive cells in thelymphatic or vascular endothelium divided by total Prox1 or DAPI-stainednuclei in each comparable field.

In situ hybridizationIn situ hybridization was performed on paraffin sections of 6 mm withradiolabeled single-stranded RNA probes. Probes were 0.42 kb mouse Tie1fragments corresponding to nucleotides 2221-2639 of NM_011587.2obtained by RT-PCR on mouse E18.5 lung RNA.

Quantitative RT-PCR, northern analysis and western blottingRNA from the lungs of wild-type littermates, Tie1+/neo and Tie1neo/neo

embryos at E18.5 was isolated using TRIzol Reagent (Invitrogen) withadditional DNase treatment (Promega). cDNA was then generated using theSuperScript II Reverse Transcriptase Kit (Invitrogen). Quantitative PCR wasperformed on the LightCycler machine (Roche) using the LightCycler DNAMaster SYBR Green I Kit (Roche) and rodent Gapdh control reagents(Applied Biosystems). Sequences for the mouse Tie1 primers have beenpublished (Taichman et al., 2003). All assays were repeated at least twiceand all samples were run in triplicate.

For northern analysis, RNA was hybridized with a probe based on thesame 0.42 kb mouse Tie1 fragment used for in situ hybridization followinggel electrophoresis and blots were stripped and rehybridized with a mouseglyceraldehyde-3-phosphate dehydrogenase (G3pdh) cDNA probe.

Western blotting was performed using standard protocols fromhomogenized E18.5 lungs using primary antibody (rabbit anti-Tie1, SantaCruz Biotechnology) detected with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit, Molecular Probes) and imaged with the Odysseyinfrared scanner. Mouse b-actin was used as a control.

LymphangiographyTo visualize functional lymphatic vessels, FITC-dextran (Sigma, MW=2000kDa, 8 mg/ml in PBS) was injected intradermally into the back of theembryonic forelimb at E17.5 and E18.5 as previously described (Hirashimaet al., 2008). Lymphatic flow carrying FITC-dextran in embryos wasanalyzed by Olympus fluorescence microscopy. For quantitative analysis oflymphangiography, the total length of lymphatic vessels carrying dye inembryos at E17.5 and E18.5 was measured using ImageJ software based onthe photos taken after 2 minutes of injection.

Morphometric analysisThe relative lymph sac size was quantified as described previously (Fritz-Six et al., 2008) using both Lyve1-stained and Hematoxylin and Eosin(H&E)-stained paraffin sections. Briefly, transverse sections of the jugularregion of mutant and wild-type embryos were imaged on an OlympusSZX16 dissecting microscope. Sections containing jugular lymph sacs thatwere matched for the same anteroposterior level were blindly measuredusing ImageJ software. In order to normalize for section variability, the areaof the lymph sac was divided by the area of the adjacent jugular vein.

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Quantitative analysis of dermal lymphatic parameters (vessel diameter,vessel density and total vessel area per field) of wild-type, Tie1neo/neo andTie1–/– embryos at E13.5 to E17.5 was performed as described (François etal., 2008), based on Vegfr3/Lyve1 wholemount immunostaining.

Data are expressed as the means ± standard deviation. Statistical analysiswas conducted using the two-tailed Student’s t-test. A P-value <0.05 wasconsidered significant.

RESULTSTie1 is expressed in the lymphatic vasculaturethroughout embryonic developmentWe examined Tie1 expression at E10.5-E11.5 by X-gal staining ofTie1+/lacZ mice (Puri et al., 1995) and immunostaining with Prox1antibody. The Tie1+/lacZ mice carry a b-gal reporter cassette insertedinto exon 1 of the Tie1 locus under the control of the Tie1 promoter.This insertion allowed us to use b-gal detection to precisely followTie1 expression throughout development. We detected that Tie1 isco-expressed with Prox1 in LECs at all stages examined. As seen inFig. 1A-C, a few endothelial cells lining the wall of the anteriorcardinal vein were Prox1-positive. Some endothelial cells buddingfrom the anterior cardinal vein were also Prox1-positive, which werelocated toward the outer margin of the vein. These cells representeda subpopulation of endothelial cells – the LECs. Almost all of theseProx1-positive cells co-expressed Tie1 (X-gal positive). The rest ofthe X-gal-positive/Prox1-negative cells corresponded to ECs of thevascular system.

Next, we determined whether Tie1 is expressed in LECs at laterstages. As the first detectable lymphatic vessels (jugular lymphsacs) in the mouse embryo can be morphologically distinguishedfrom the blood vessels at E13.5, we examined Tie1 expression atE13.5 by X-gal staining of Tie1+/lacZ mice. As seen in Fig. 1D-E, atE13.5, X-gal (Tie1) stains not only the arteries, veins and bloodcapillaries, but also lymphatics, as judged initially by theiranatomical position: in the neck region, the major lymphatic vesselsare formed in close proximity to the cardinal vein. To preciselydetermine whether these vessels were lymphatics, we performedimmunolabeling with antibody against Vegfr3, which is largelyrestricted to LEC after mid-gestation (Kaipainen et al., 1995). Wedetected strong Vegfr3 expression in the jugular lymphatic vessels(Fig. 1E). Arteries did not express Vegfr3 but were clearly X-gal-positive. Veins were X-gal-positive but Vegfr3-negative. Therefore,Tie1 is expressed in the lymphatic endothelium duringembryogenesis at E13.5. The expression of Tie1 in the lymphaticvessels at E13.5 was further confirmed by in situ hybridization (Fig.1F).

To determine whether Tie1 expression continues in lymphaticvessels throughout development, we examined skin, intestine,mesentery and lung (Fig. 1G-J) from E17.5 embryos. As observedat earlier stages, X-gal staining in ECs colocalized with antibodiesagainst Vegfr3 or Lyve1 (data not shown). Thus, Tie1 is expressedin the Prox1-positive venous LEC progenitors and lymphaticendothelia throughout embryonic development.

Characterization of a hypomorphic Tie1 alleleTo circumvent the embryonic and perinatal lethality observed inhomozygous-null mutant mice (Puri et al., 1995; Sato et al., 1995),we created a conditional allele of mouse Tie1 gene (see Fig. S1 inthe supplementary material). Once the neomycin selection cassettewas removed, the levels of Tiel expression in Tie1loxP/loxP mice wereindistinguishable from those of wild-type animals. When these micewere crossed with E2A-Cre transgenic mice, which express Crerecombinase in the germ line (Lakso et al., 1996), homozygous-

deleted pups (Tie1–/–) died of hemorrhage and severe edema in uteroor at birth, displaying the same phenotypes of the conventionalknockout mice (Sato et al., 1995).

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Fig. 1. Tie1 expression in the LECs during embryogenesis.(A-C) Transverse frozen section of E11.5 Tie1+/lacZ mouse embryosdouble-labeled with X-gal (blue staining in A and C) and Prox1antibody (red in B and C). The polarized lymphatic endothelial cells(ECs) lining the wall of the anterior cardinal vein (cv) and budding fromthe anterior cv were labeled by Prox1 and were also X-gal-positive,indicating the co-expression of Prox1 and Tie1. X-gal-positive/Prox1-negative cells corresponded to ECs of the vascular system includingcapillaries and arteries (a). (D,E) Transverse frozen section through thejugular region of an E13.5 Tie1+/lacZ embryo double-labeled with X-gal(D, blue) and Vegfr3 antibody (E, red). The Tie1-expressing cells wereconfirmed to be jugular lymphatic sacs (jls) by Vegfr3 labeling. X-gal-positive/Vegfr3-negative cells corresponded to ECs of the non-lymphatic vascular system. (F) Transverse section through the neck of anE13.5 wild-type embryo hybridized with a murine Tie1 antisenseriboprobe. Tie1 mRNA (red signal) is expressed in all ECs.(G-J) Transverse frozen sections of skin (G), intestine (H), mesentery (I)and lung (J) from E17.5 Tie1+/lacZ embryos double-labeled with X-gal(blue) and Vegfr3 antibody (red). Tie1 is not only expressed in bloodvessels and capillaries (X-gal-positive only), but also co-expressed withVegfr3 in the lymphatics (X-gal- and Vegfr3-positive, white arrows).

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Interestingly, when the neomycin selection cassette used in theinitial targeting construct was not removed, the resultant Tie1neo

allele appeared to be hypomorphic. Tie1+/neo mice were bred toobtain homozygous pups, and mice homozygous for this allele(Tie1neo/neo) displayed a more variable degree of severity thandetected in the null mutant animals. Before E16.5, the majority(99/108, 91.7%) of the hypomorphic homozygous embryosexhibited mild to severe edema without signs of hemorrhage (Fig.2B,D). By contrast, all of the Tie1–/– mutant embryos with the samebackground manifested severe edema and 16.7% (4/24) alsodisplayed localized hemorrhages distributed throughout the bodysurface. In Tie1–/– mutant embryos, the hemorrhagic defect wasusually observed from E13.5 onward (Fig. 2C), occasionally evenas early as E13.0 (data not shown). After E16.5, 34.6% (9/26) ofhypomorphic embryos also developed hemorrhage but it waslocalized to the tip of the tail, and occasionally (4/26) also at the tipsof the toes (Fig. 2E). By contrast, the vast majority (92.5%) of the

null mutant Tie1–/– embryos exhibited extensive hemorrhage fromE16.5 (Fig. 2F) to E18.5 or embryonic demise (Fig. 2G). Rarehypomorphic homozygous embryos died before E18.5 and a few(7 mice from 28 litters) survived to adulthood.

To determine if Tie1 expression in the hypomorphic embryos wasdifferent from that in wild-type embryos, we performed northernblot analysis using embryonic lungs, a site of robust Tie1 expressionduring embryogenesis (Taichman et al., 2003). As seen in Fig. 2H,Tie1 mRNA level is reduced to about half of that detected in wild-type embryos, whereas only a weak band was detected inhomozygous mutant lungs. We measured Tie1 mRNA byquantitative PCR analysis of the same samples, which showed thatthe Tie1 mRNA expression in the E18.5 lungs of Tie1+/neo andTie1neo/neo embryos was reduced by 50.4% and 79.4%, respectively(Fig. 2I). Consistently, western blot analysis showed that Tie1protein levels in the E18.5 lungs of Tie1+/neo and Tie1neo/neo embryoswere reduced by 43.8% and 76.0%, respectively (Fig. 2J).

Tie1 attenuation results in enlarged lymph sacsWe next examined sequential stages of lymphatic development inwild-type, Tie1-hypomorphic and Tie1-null mutant embryos.Histology of Tie1neo/neo and Tie1–/– embryos confirmed the presenceof extensive interstitial edema (Fig. 3C,E). Lymph sacs wereidentified in wild-type, Tie1neo/neo and Tie1–/– embryos at E13.5 byimmunohistochemical recognition of the lymphatic-specific makersLyve1 (Fig. 3B,D,F) and Vegfr3 (data not shown), indicating normallymphatic differentiation from venous vasculature. However, weobserved that the jugular lymph sacs of Tie1neo/neo and Tie1–/–

embryos appeared strikingly larger than those of litter-matched wild-type embryos. Computerized morphometry was used to calculatelymph sac area. At E13.5, the jugular lymphatic sacs in the wild-typeembryos are located lateral to the internal jugular vein and dilate toa maximal diameter of approximately 2-3 times the size of thediameter of the internal jugular vein (Fig. 3A,B) (Gittenberger-DeGroot et al., 2004). In Tie1neo/neo and Tie1–/– embryos at E13.5, themaximal diameter of the jugular lymphatic sacs was approximately10 times the size of the diameter of the internal jugular vein (Fig.3D,F,G). This phenotype persists until at least E15.5 (data notshown).

Tie1 attenuation leads to an early increase inproliferation of LECsTo explore why the lymph sacs were dilated, we used a BrdUincorporation assay to assess proliferating cells in both lymph sacsand adjoining jugular veins. At E13.5, we observed no difference inthe percentage of proliferative Prox1-positive ECs in lymphatic sacs(27.1±4.9% versus 25.6±4.1%) or jugular veins (data not shown)between the hypomorphic and wild-type embryos. However, whenembryos at E11.5 and at E12.5 were compared, the percentage ofproliferative Prox1-positive cells in Tie1neo/neo embryos at E11.5 andE12.5 was significantly higher than that in wild-type littermates(P<0.05), by 17.9% and 22.2%, respectively (Fig. 4). This increaseof proliferative LECs was confirmed by co-labeling of adjacentsections with Prox1 and Ki67 (Fig. 4A,B).

We then quantified the total number of LECs in the cardinal veinand jugular lymph sac areas of embryos at sequential stages oflymphatic development. At E10.5, the number of Prox1-positiveLECs in Tie1neo/neo (Fig. 5B,J) and Tie1–/– (Fig. 5C,J) embryos wereincreased by 23.2% and 29.1%, respectively (Fig. 5A). Similarly,at E11.5, the number of LECs in Tie1neo/neo (Fig. 5E,J) and Tie1–/–

(Fig. 5F,J) embryos were increased by 35.0% and 38.9%,respectively, and at E12.5, the number of LECs in Tie1neo/neo (Fig.

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Fig. 2. Characterization of the Tie1 hypomorphic allele. (A) A wild-type embryo at E13.5. (B-D) Hypomorphic Tie1 homozygous embryos(neo/neo) demonstrating dorsal subcutaneous edema at E13.5 (B) andE14.5 (D), and a Tie1 homozygous-null mutant embryo (–/–) at E13.5(C) showing hemorrhage and dorsal edema. (E-G) A hypomorphic Tie1homozygous embryo displaying hemorrhage only at the tip of the tailand the tips of the toes at E16.5 (E) in contrast to the Tie1homozygous-null mutant embryos which demonstrated severehemorrhage (F) or death (G) at E16.5. (H) Northern blot analysis of Tie1mRNA expression level in the E18.5 lungs of wild-type, Tie1+/neo andTie1neo/neo embryos. (I) Real-time RT-PCR showing that compared withwild-type embryos (+/+), Tie1 mRNA expression levels in the E18.5lungs of Tie1+/neo (+/neo) and Tie1neo/neo (neo/neo) embryos werereduced to 49.6% and 20.6%, respectively. (J) Western blot analysis ofTie1 expression level in the E18.5 lungs of the three genotypesexamined.

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5H,J) and Tie1–/– (Fig. 5I,J) embryos were increased by 44.2% and53.5%, respectively. These results suggest that an increasedproduction of LECs at the initiation of lymphangiogenesiscontributes to the dilated lymph sacs seen in the Tie1 hypomorphicand mutant mice.

To evaluate whether Tie1 attenuation resulted in a global defectin endothelial differentiation, we used dual immunoflourescence atE11.5 (Fig. 5D-F) and E12.5 (see Fig. S2 in the supplementarymaterial) with specific vascular EC markers – Pecam1, CD34,Icam1, endoglin or VE-cadherin and Prox1. We observed nodifference among wild-type, Tie1neo/neo and Tie1–/– embryos in theexpression of these five markers in vascular endothelium or at thedorsal part of the cardinal vein, the site where lymphatic ECdifferentiation occurs during embryogenesis. Thus, the abnormaldilated lymph sac defects in Tie1neo/neo and Tie1–/– embryos couldnot be ascribed to a primary endothelial defect.

Tie1 attenuation leads to an increase in lymphaticendothelial cell apoptosisTo determine whether the abnormal lymphatic patterning ofTie1neo/neo mice could also be associated with abnormal ECapoptosis, we used TUNEL detection in conjunction with Vegfr3labeling and DAPI nuclear localization to identify apoptosis. Wedetected no significant difference in the percentage of apoptoticLECs in the wall of the cardinal vein and jugular lymph sac areasbetween wild-type (E11.5, 3.6±0.5%; E12.5, 3.2±0.8%) andTie1neo/neo (E11.5, 3.4±0.9%; E12.5, 2.9±1.1%) embryos (see Fig.S3 in the supplementary material). However, at E13.5, weidentified an increase in apoptosis in LECs of hypomorphicembryos. Only 2.5±0.6% TUNEL-positive cells were detected inthe lymphatic endothelium of the wild-type jugular lymphatic sacs(Fig. 6A-C,G), whereas we observed 5.7±0.9% TUNEL-positiveLECs in the corresponding area of hypomorphic embryos (Fig.6D-F,G). Interestingly, almost no EC apoptosis was seen in thejugular vein or arteries of wild-type or hypomorphic embryos.Taken together, these results demonstrate that reduction of Tie1during embryonic development leads to a specific increase inlymphatic, but not venous, EC apoptosis at later stages oflymphangeogenesis.

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Fig. 3. Tie1 attenuation leads to dilation of jugular lymph sacs atE13.5. (A-F) Transverse sections through the jugular region of wild-typelittermates (A,B), Tie1neo/neo (C,D) and Tie1–/– (E,F) embryos at E13.5,labeled with Lyve1. (B,D,F) Higher magnification of the jugular region(black triangle) in A, C and E, respectively. Note the remarkable increasein size of lymph sacs relative to the jugular vein and carotid artery in thehypomorphic (D) and mutant (F) embryos and a thickened dermis andsubcutaneous layer (double-headed arrows) in the hypomorphic (C) andmutant (E) embryonic skin when compared with the wild-typelittermates (A). (G) Quantification of lymph sac area, normalized tojugular vein area in hypomorphic embryos, mutant embryos and theirwild-type littermates at E13.5. n>6 embryos per genotype. *, P<0.05.jls, jugular lymph sacs; jv, jugular vein. Scale bars: 200mm.

Fig. 4. Tie1 attenuation during early embryonic developmentleads to an increase in LEC proliferation. (A-D) Transverse sectionsof wild-type littermates (A,C) and Tie1neo/neo (B,D) embryos through thejugular region at E11.5 (A,B) and E12.5 (C,D) were immunolabeled forProx1 (red) and Ki67 (green) or Prox1 (red) and BrdU (green). Arrowsindicate representative proliferative Prox1-positive lymphatic endothelialnuclei in the wall of the cardinal vein and jugular lymph sac areas. cv,cardinal vein; ls, lymph sac. Scale bar: 100mm. (E) The percentage ofproliferative Prox1-positive cells in Tie1neo/neo embryos (gray bars) wassignificantly higher than that in wild-type littermates at E11.5 and atE12.5 (*, P<0.05), but no difference was seen at E13.5. Resultsrepresent the means ± the s.d. of 5 embryos per group.

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Abnormal dermal lymphatic pattern in Tie1mutant embryosWe next performed wholemount immunohistochemistry to examinethe development of dermal lymphatic vasculature in Tie1neo/neo

embryos at E13.5. In wild-type embryos, a few, but clear, Vegfr3-positive lymphatic vessels (Fig. 7A) and an extensive network of

lymphatic capillaries (Fig. 7D) was detected in the head dermis nearthe developing ear and in limb skin. In the Tie1neo/neo embryos,however, the lymphatic capillaries in the corresponding regionswere dilated and disorganized (Fig. 7B,E). The lymphatic defects inthe null mutant embryos (Fig. 7C,F) were more severe comparedwith those in the hypomorphic embryos. Importantly, wholemountstaining of E13.5 forelimb skin for Pecam1 revealed a relativelynormal blood vessel pattern in the hypomorphic embryos and onlymildly dilated blood vessels in the null embryos (Fig. 7G-I),indicating a preferential effect on lymphatic vessels. These resultssuggested that the severity of lymphatic defects in Tie1 mutantembryos is dependent on the dosage of Tie1 and that thedevelopment of lymphatic vasculature is more sensitive to Tie1reduction than the development of blood vasculature.

To further investigate network formation of blood vessels andlymphatic vessels, we performed wholemount double-fluorescenceconfocal microscopy of embryonic dorsal skin at later stages ofdevelopment. As seen in Fig. S4 in the supplementary material, atE14.5, the Vegfr3-positive lymphatic capillary network was detectedin wild-type control embryos, whereas lymphatic vessels weredisorganized and mildly dilated in Tie1neo/neo embryos; at E15.5,lymphatic vessels in Tie1neo/neo embryos were dilated and lymphaticvessels started to regress in some areas, and compared with thephenotype in Tie1neo/neo embryos, the lymphatic defects in Tie1–/–

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Fig. 5. Tie1 attenuation leads to an increased lymphaticendothelial cell production. (A-I) Transverse sections at E10.5 (A-C)and at E11.5 (D-F) were stained with Prox1 (red) and Pecam1 (green).E12.5 sections (G-I) were labeled with Prox1 (red) and Vegfr3 (green).Representative images show more Prox1-positive LECs (arrows in A-C)in the cardinal vein and jugular lymph sac areas in Tie1neo/neo (B,E,H)and Tie1–/– (C,F,I) embryos than in wild-type embryos (A,D,G).Compared with wild-type control, both Tie1neo/neo and Tie1–/– embryosexhibit dilated jugular lymph sacs at E11.5 and at E12.5. cv, cardinalvein; da, dorsal aorta; ls, lymph sac. Scale bars: 100mm. Note: thelymphatic marker Vegfr3 has weak expression in blood vessels at E12.5.(J) Quantitative analysis of the number of Prox1-positive nuclei in thecardinal vein and jugular lymph sac areas of embryos at E10.5 to E12.5.*, P<0.05.

Fig. 6. Tie1 attenuation during embryonic development leadsto an increase in lymphatic endothelial cell apoptosis.(A-F) Transverse sections of wild-type littermates (A-C) and Tie1neo/neo

(D-F) embryos through the jugular region at E13.5 were immunolabeledfor Vegfr3 (red) and TUNEL (green). (G) The percentage ofVegfr3/TUNEL-positive apoptotic cells of the jugular lymph sacs wassignificantly higher in Tie1neo/neo embryos compared with wild-typelittermates (*, P<0.05). Arrows indicate apoptotic LECs. cv, cardinalvein; jls, jugular lymph sac. Scale bar: 100mm.

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embryos were even more severe. This was confirmed by Lyve1immunoreactivity of skin from the three genotypes at E15.5 (see Fig.S5 in the supplementary material).

By E16.5, the severity of dermal lymphatic defects in Tie1hypomorphic embryos was even more evident: Vegfr3-positivelymphatic vessels were dramatically dilated in some areas orseverely disrupted in other areas (see Fig. S6A-C in thesupplementary material). At E17.5 (Fig. 9; see Fig. S6D-F in thesupplementary material), the Lyve1-positive lymphatic networkswere dense and well organized in wild-type embryos but weremarkedly dilated, disorganized and irregular in Tie1 hypomorphicembryo lymphatics. In addition, most of the wild-type lymphaticcapillaries were interconnected and only a few blind beginninglymphatic capillaries were detected. By contrast, the formation oflymphatic capillary networks was impaired in the head skin of thehypomorphic mice. The skin in the hypomorphic embryos exhibitedincomplete network patterning with an increased number of blindbeginnings of lymphatics. Some regions were largely bereft oflymphatics (Fig. 8B,F; see Fig. S5 and Fig. S6 in the supplementarymaterial), indicating regression of the developing lymphatic vessels.

To quantify the effect of Tie1 attenuation on lymphatic vesselprofiles, we measured lymphatic vessel area, density and diameter(Fig. 8M-O). These studies documented an increase in vessel

diameter in the hypomorphic and mutant embryos at alldevelopmental time points examined. The dilated dermal lymphaticsseen in the Tie1 hypomorphic and mutant mice could be ascribed tothe abnormally high production of LECs early in development,rather than an increase in LEC size. The total number of LECs(Prox1-positive nuclei) in each dilated dermal lymphatic vessel washigher in Tie1neo/neo embryos than in wild-type embryos. Thisincrease in LEC number was associated with an increase inlymphatic vessel area such that total LEC density (Prox1-positivenuclei/lymphatic vascular area) was not significantly altered inmutant embryos (see Fig. S7 in the supplementary material).However, density of lymphatic vessels (lymphatic vessel area/field)in the hypomorphic and mutant embryos was reduced from E15.5,indicating vessel regression. The total vessel area in thehypomorphic and mutant embryos was significantly higher owingto vessel dilation at E13.5, but started to decrease compared withwild-type controls from E15.5 owing to vessel disruption orregression. Similar to the jugular lymphatic sacs, we also detectedaccentuated Vegfr3-positive EC apoptosis in the dermis of thehypomorphic embryos at E15.5 and at E17.5 with both TUNELdetection and antibodies against cleaved caspase 3, with minimalVegfr3-positive EC apoptosis detected in wild-type embryos (seeFig. S8 in the supplementary material).

By contrast, blood vascular patterning was relatively unaffectedin the hypomorphic embryos at E14.5, E15.5 (data not shown),E16.5 (see Fig. S6A-C in the supplementary material) or E17.5 (Fig.8C-L). Thus, attenuation in Tie1 was sufficient to alter the patterningof the lymphatic vasculature without affecting the non-lymphaticvasculature, suggesting that lymphatic dysfunction contributes to theedema seen in the Tie1 hypomorphic and mutant mice. This findingalso supports the hypothesis that developing lymphatics are moresensitive to Tie1 reduction than the arterial or venous vasculature.

Lymphatic function is impaired in Tie1neo/neo

embryosTo assess whether structural defects seen in dermal lymphaticvessels lead to impaired lymphatic drainage, we evaluated lymphaticfunction by injecting high-molecular-weight FITC-dextran intoembryonic limbs. Lymphangiography showed that dye uptake wasrapid in the collecting lymphatic vessels and network of capillariesin wild-type mice at E17.5 (Fig. 9A). However, the transportfunction of lymphatic vessels in the hypomorphic embryos wasimpaired, consistent with the severity of the subcutaneous edemadefect. As seen in Fig. 9B, FITC-dextran labeled only a fewdisorganized lymphatic capillaries in the hypomorphic embryo thathad severe edema and localized hemorrhages at E17.5, indicatingattenuation and/or poor function of the lymphatic capillary network.We obtained similar results using wild-type and hypomorphicembryos at E18.5 (Fig. 9C).

Lymphatic patterning is abnormal in internalorgans of Tie1 hypomorphic embryosIt is known that lymphatic capillaries progressively cover the heartsurface and the diaphragm from E15 onwards (Yuan et al., 2002).Wholemount staining of these hearts with Lyve1 or Vegfr3 antibodyrevealed regression of the developing lymphatic vessels at thesurface of the heart in the hypomorphic embryos compared withcontrol littermates (see Fig. S9A-F in the supplementary material),which was first observed at E16.5. The epicardial lymphatic vesselsin the hypomorphic embryos formed a primitive continuous networkbut were thinner than those in wild-type control embryos. Inaddition, the lymphatic vessels of the mutant embryos had begun to

1291RESEARCH ARTICLETie1 and lymphatic development

Fig. 7. The severity of the dermal lymphatic defects in Tie1mutant embryos at E13.5 is dosage-dependent. (A-F) Heads (A-C)and forelimbs (D-F) of wild-type (A,D), Tie1neo/neo (B,E) and Tie1–/– (C,F)embryos at E13.5 labeled with Vegfr3 antibody. Developing lymphaticnetwork in the skin around the ear is normal in wild-type embryos (A),but dilated and disorganized in Tie1neo/neo embryos (B), and even moredisrupted in Tie1–/– embryos (C). Similarly, a normal network oflymphatic capillaries was detected in the forelimb skin of wild-typeembryos (D) and an irregular lymphatic network with dilated anddisorganized lymphatics was observed in the corresponding regions ofTie1neo/neo (E) and Tie1–/– (F) embryos, respectively. (G-I) Forelimbs ofwild-type (G), Tie1neo/neo (H) and Tie1–/– (I) embryos at E13.5 werelabeled with Pecam1 antibody and the blood vessel pattern appearednormal. Arrowheads indicate the diameter of the lymphatic vessels.Scale bars: 250mm.

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regress in some areas. At E17.5, the entire lymphatic network wasdisrupted in the hypomorphic embryos and by E18.5, only a fewsingle disrupted lymphatic vessels were detected on the surface ofthe heart. In the central portion of the diaphragm at E16.5, some ofthe larger collecting lymphatics in the hypomorphic embryos weredilated and disorganized compared with wild-type embryos (see Fig.S9G-L in the supplementary material). By E17.5 and E18.5, therewas a striking reduction in the number of lymphatics in the wholediaphragm of the hypomorphic embryos. Furthermore, all of thelymphatics were much thinner than those in wild-type controlembryos and the network was disrupted, indicating regression of thedeveloping lymphatics over the entire diaphragmatic surface. Takentogether, the Tie1neo/neo mice demonstrated abnormally patternedlymphatic vessels in all internal organs examined.

DISCUSSIONGenetic studies in mice have demonstrated that Tie1 is required fordevelopment and maintenance of the vascular system. Deletion ofTie1 leads to embryonic lethality due to edema, hemorrhage andmicrovessel rupture (Puri et al., 1995; Sato et al., 1995).Interestingly, the timing of previously reported embryonic demiseat E13.5 correlated with the earliest time when lymphatic vessels canbe morphologically distinguished from the blood vessels. Theprevious report (Sato et al., 1995) and our study showed that the firstvisible defect in the Tie1 mutants is dorsal subcutaneous edema fromE13.5 onward. In this report, we have documented that Tie1 isexpressed in LEC progenitors at E10.5 and E11.5 before lymphaticvessels are formed. This expression of Tie1 in the lymphaticendothelium is maintained throughout embryonic development and

persisted in the postnatal animal. The early expression in thelymphatic endothelium suggests a unique role for Tie1 in thedevelopment and function of the lymphatic vessels.

The intronic insertion of a neo cassette causes aberrant splicing ofTie1 transcripts, reducing the amount of mRNA encoding functionalTie1 protein to ~20% of normal levels. This Tie1 hypomorphic alleleenabled us to demonstrate that a dosage-dependent reduction of Tie1results in progressive defects in lymphatic patterning not seen in otherEC populations. Tie1 attenuation resulted in significantly enlargedjugular lymphatic vessels. Consistent with structural defects seen inthe lymphatics, lymphatic function was also impaired in hypomorphicembryonic skin. We detected no apparent lymphatic defects in theheterozygous Tie1+/neo embryos at any developmental stage.

Interestingly, although the Tie1 hypomorphic embryos displaysevere generalized edema, there was almost no apparent hemorrhageduring the entire period of embryonic development. A few embryosdeveloped hemorrhage at the tips of the tail or digits and thisoccurred at a much later time point than the diffuse hemorrhageobserved in the complete null embryos. In addition, althoughlymphatic defects were obvious in most of the hypomorphicembryos, the pattern of blood vessels appeared relatively normal. Infact, when we examined expression of the specific blood vascularEC markers in the major blood vessels and in particular at the dorsalpart of the cardinal vein, the site where lymphatic EC differentiationoccurs during the initiation of lymphangiogenesis, we could detectno differences in the pattern of expression between wild-type andhypomorphic mutant embryos. These results further suggest that thedevelopment of lymphatic vasculature is more sensitive to Tie1reduction than the development of blood vasculature.

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Fig. 8. Dermal lymphatic defectsresulting from Tie1 attenuation.(A-L) Head skin of wild-type littermates(A,C,E,G,I,K) and Tie1neo/neo (B,D,F,H,J,L)embryos were labeled with antibodiesagainst Lyve1 (red) and Pecam1 (green) atE17.5. In comparison with control embryos,lymphatic networks in the skin ofhypomorphic embryos were remarkablydilated, disorganized and regressed, but thenon-lymphatic blood vessel patternappeared normal. The arrow indicatesregressing lymphatics. Arrowheads indicatethe diameter of the lymphatic vessels.Original magnification of confocal images:�10 (A-F); �40 (G-L). Scale bars: 100mm.(M-O) Quantitative analysis of lymphaticparameters in the skin of wild-type,Tie1neo/neo and Tie1–/– embryos at E13.5 toE17.5 based on Vegfr3/Lyve1 wholemountimmunostaining. (M) Vessel size (diameter).(N) Vessel density. (O) Total vessel area perfield. *, P<0.05.

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It is commonly thought that most of the lymphovascular systemin mice is of venous origin (Oliver, 2004; Oliver and Alitalo, 2005).However, recent data argues for a dual origin of LECs in the mousewith incorporation of mesenchymal lymphangioblast-derived cellsinto the lining of lymph vessels during development providing aminor contribution to the lymphatics (Buttler et al., 2006; Buttler etal., 2008). Although our work does not directly address whether alllymphatics are of venous origin or whether there is an additionalcontribution from a mesenchymal cell population, the fact that weobserved similar abnormalities in both the central lymphatics as wellas in the dermis of the embryo would suggest that Tie1 is requiredfor the normal development of both the central lymphatics and theperipheral lymphatics.

Jugular lymphatic sac dilation was a prominent feature in the Tie1mutant embryos. Attenuation of Tie1 leads to an increased LECproduction before mid-gestation in the Tie1 hypomorphic andmutant mice. Consistently, we detected significantly moreproliferative LECs in the cardinal vein and jugular lymph sac areasof Tie1 hypomorphic and mutant embryos at E11.5 and E12.5, whenspecification of lymphatic vasculature occurs (Francois et al., 2008).Although the proliferation rate of LECs in jugular lymphatic sacs ofTie1 hypomorphic and mutant embryos at E13.5 returns to normal,the absolute total number of proliferative LECs is still higher thanthat in wild-type embryos because the absolute number of LECs ishigher (Fig. 5J).

This early period of increased LEC proliferation was followedby an accentuation of apoptosis in the latter stages of lymphaticvascular development. We found normal LEC apoptosis in Tie1hypomorphic and mutant embryos at early stages (E11.5 andE12.5) but a selective increase in LEC apoptosis from E13.5. Thisis consistent with the observed regression of the already-formeddeveloping lymphatics and the previous report that Tie1 inhibitsapoptosis and is required for EC survival (Kontos, et al., 2002).From these findings, we conclude that attenuation of Tie1 inlymphatics results in dilation due to an elevated production ofLECs during specification of lymphatic vasculature, andregression after mid-gestation due to increased apoptosis of LECs.This process leads to a disorganized and disrupted lymphaticnetwork.

The lymphatic defects of Tie1 mutant mice are reminiscent ofthe lymphatic phenotypes of the Tie2 ligand, Ang2, mutant mice,although the latter exhibits only dilated lymphatic vessels withoutregression and with no defects in the size of jugular lymphatic sacs(Gale et al., 2002; Dellinger et al., 2008). Ang2 is involved in theremodeling and stabilization of lymphatic vessels. Ang2–/– miceexhibit defects in the remodeling of the blood vasculature, butsurprisingly, even more severe defects of the lymphatic system.Ang2–/– mice display chylous ascites, peripheral lymphedema andhypoplasia of the lymphatic vasculature (Gale et al., 2002).Lymphatic vessels in Ang2–/– mice fail to mature and do not exhibita collecting vessel phenotype. Furthermore, dermal lymphaticvessels in Ang2–/– pups prematurely recruit smooth muscle cellsand do not undergo proper postnatal remodeling (Dellinger et al.,2008).

To date, the Tie1 receptor remains primarily an orphan receptor,as no ligand for Tie1 has been identified and only ligand-independent signal transduction pathways have been characterized(McCarthy et al., 1999; Yabkowitz et al., 1999). However, Tie1phosphorylation can be induced by overexpression of multipleangiopoietin proteins (Ang1 and Ang4) in vitro and activation isamplified via association with Tie2 (Saharinen et al., 2005). Tie1 andTie2 receptor heterodimers had been shown to exist in cultured ECs(Marron et al., 2000) but the cellular function of heterodimerizationremains to be determined. As Tie1 is expressed in the endotheliumof the same lymphatics as Ang2 during embryogenesis andenhanced Tie1 mRNA expression occurs together with Ang2 mRNAin the adults (Gale et al., 2002; Iljin et al., 2002), it is tempting tospeculate that Tie1 might be modulating Ang2 function viainteraction with the Tie2 receptor in the lymphatic ECs as has beendocumented in cultured ECs (Yuan et al., 2007) and endothelialprogenitor cells (Kim et al., 2006). Interestingly, investigators haverecently shown that Vegf can cause a 4-fold increase inphosphorylation of Tie2 that is independent of angiopoietinexpression but dependent on proteolytic cleavage of Tie1 andsubsequent transphosphorylation of Tie2, further supporting thepossibility of Tie1 modulation of Tie2 signaling (Singh et al., 2009).

However, the expression of Tie2 and its role in LEC developmenthas not been well defined. Adult Lyve1-positive lymphaticvessels have previously been shown to express Tie2 byimmunofluorescence labeling of mouse ear skin and the smallintestine using an antibody against Tie2 (Morisada et al., 2005;Tammela et al., 2005). However, other groups failed to detect GFPexpression by lymphatic vessels in the adult (Dellinger et al., 2008)or embryos (Srinivasan et al., 2007) from Tie2-GFP transgenic miceusing either immunohistochemical (GFP) analyses or in situhybridization. One possible explanation for this discrepancy is thatthe regulatory elements controlling the expression of Tie2 by

1293RESEARCH ARTICLETie1 and lymphatic development

Fig. 9. Tie1 attenuation leads to impaired lymphatic drainagefunction. (A,B) Representative image of the subcutaneous collectinglymphatic vessels (arrows) and lymphatic capillaries (arrowheads) usingfluorescence microscopy after intradermal FITC-dextran injection intoembryonic forelimbs at E17.5. Sites of injection are indicated by dashedcircles. The lymphatic network in Tie1neo/neo embryos (B) is disorganizedand uptake of FITC-dextran is attenuated when compared with thatseen in wild-type mice (A). Scale bar: 500mm. (C) Quantitative analysisof the total length of lymphatic vessels (2 minutes after each injection)in wild-type versus Tie1neo/neo embryos at E17.5 and at E18.5.*, P<0.05.

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lymphatic vessels are not included in the Tie2-GFP transgenicconstruct. Further delineation of the expression pattern of Tie2should resolve this discrepancy.

In summary, it is now evident that the Tie receptors and theirligands demonstrate context-dependent regulation of vascularremodeling (Eklund et al., 2006) and our work suggests a uniquedosage-dependent role for Tie1 in lymphatic ontogeny. WhetherTie1 signals independently or in association with Tie2 is a focus ofongoing investigation. However, the development of ahypomorphic, conditional Tie1 allele in combination with theavailable lymphatic-specific Cre deletor lines (Srinivasan et al.,2007) should provide the necessary reagents for the temporal- andlymphatic-specific deletion required for further investigation.

AcknowledgementsWe thank Dr Lawrence Price (Vanderbilt University) for providing the antibodiesagainst Prox1 and valuable discussions and Dr Christopher Brown (VanderbiltUniversity) for critical reading of the manuscript. This work was supported byNIH grants R01 HL086964 (S.B.) and DK038517 and a grant-in-aid from theMarch of Dimes. Deposited in PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.043380/-/DC1

ReferencesAyadi, A., Zheng, H., Sobieszczuk, P., Buchwalter, G., Moerman, P., Alitalo,

K. and Wasylyk, B. (2001). Net-targeted mutant mice develop a vascularphenotype and up-regulate egr-1. EMBO J. 20, 5139-5152.

Buttler, K., Kreysing, A., von Kaisenberg, C. S., Schweigerer, L., Gale, N.,Papoutsi, M. and Wilting, J. (2006). Mesenchymal cells with leukocyte andlymphendothelial characteristics in murine embryos. Dev. Dyn. 235, 1554-1562.

Buttler, K., Ezaki, T. and Wilting, J. (2008). Proliferating mesodermal cells inmurine embryos exhibiting macrophage and lymphendothelial characteristics.BMC Dev. Biol. 8, 43.

Dellinger, M., Hunter, R., Bernas, M., Gale, N., Yancopoulos, G., Erickson, R.and Witte, M. (2008). Defective remodeling and maturation of the lymphaticvasculature in Angiopoietin-2 deficient mice. Dev. Biol. 319, 309-320.

Dumont, D. J., Fong, G. H., Puri, M. C., Gradwohl, G, Alitalo, K. andBreitman, M. L. (1995). Vascularization of the mouse embryo: a study of flk-1,tek, tie, and vascular endothelial growth factor expression during development.Dev. Dyn. 203, 80-92.

Eklund, L. and Olsen, B. R. (2006). Tie receptors and their angiopoietin ligandsare context-dependent regulators of vascular remodeling. Exp. Cell Res. 312,630-641.

François, M., Caprini, A., Hosking, B., Orsenigo, F., Wilhelm, D., Browne, C.,Paavonen, K., Karnezis, T., Shayan, R., Downes, M. et al. (2008). Sox18induces development of the lymphatic vasculature in mice. Nature 456, 643-647.

Fritz-Six, K. L., Dunworth, W. P., Li, M. and Caron, K. M. (2008).Adrenomedullin signaling is necessary for murine lymphatic vasculardevelopment. J. Clin. Invest. 118, 40-50.

Gale, N. W., Thurston, G., Hackett, S. F., Renard, R., Wang, Q., McClain, J.,Martin, C., Witte, C., Witte, M. H., Jackson, D. et al. (2002). Angiopoietin-2is required for postnatal angiogenesis and lymphatic patterning, and only thelatter role is rescued by Angiopoietin-1. Dev. Cell 3, 411-423.

Gittenberger-De Groot, A. C., Van Den Akker, N. M., Bartelings, M. M., Webb,S., Van Vugt, J. M. and Haak, M. C. (2004). Abnormal lymphatic development intrisomy 16 mouse embryos precedes nuchal edema. Dev. Dyn. 230, 378-384.

Hashiyama, M., Iwama, A., Ohshiro, K., Kurozumi, K., Yasunaga, K.,Shimizu, Y., Masuho, Y., Matsuda, I., Yamaguchi, N. and Suda, T. (1996).Predominant expression of a receptor tyrosine kinase, TIE, in hematopoietic stemcells and B cells. Blood 87, 93-101.

Hirashima, M., Sano, K., Morisada, T., Murakami, K., Rossant, J. and Suda, T.(2008). Lymphatic vessel assembly is impaired in Aspp1-deficient mouseembryos. Dev. Biol. 316, 149-159.

Hosking, B., François, M., Wilhelm, D., Orsenigo, F., Caprini, A., Svingen, T.,Tutt, D., Davidson, T., Browne, C., Dejana, E. et al. (2009). Sox7 and Sox17are strain-specific modifiers of the lymphangiogenic defects caused by Sox18dysfunction in mice. Development 136, 2385-2391.

Huang, X. Z., Wu, J. F., Ferrando, R., Lee, J. H., Wang, Y. L. et al. (2000). Fatalbilateral chylothorax in mice lacking the integrin a9b1. Mol. Cell. Biol. 20, 5208-5215.

Iljin, K., Petrova, T. V., Veikkola, T., Kumar, V., Poutanen, M. and Alitalo, K.(2002). A fluorescent Tie1 reporter allows monitoring of vascular developmentand endothelial cell isolation from transgenic mouse embryos. FASEB J. 16,1764-1774.

Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V. W., Fang, G.H., Dumont, D., Breitman, M. and Alitalo, K. (1995). Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium duringdevelopment. Proc. Natl. Acad. Sci. USA 92, 3566-3570.

Kim, K. L., Shin, I. S., Kim, J. M., Choi, J. H., Byun, J., Jeon, E. S., Suh, W. andKim, D. K. (2006). Interaction between Tie receptors modulates angiogenicactivity of angiopoietin2 in endothelial progenitor cells. Cardiovasc. Res. 72,394-402.

Kontos, C. D., Cha, E. H., York, J. D. and Peters, K. G. (2002). The endothelialreceptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt toinhibit apoptosis. Mol. Cell. Biol. 22, 1704-1713.

Korhonen, J., Polvi, A., Partanen, J. and Alitalo, K. (1994). The mouse tiereceptor tyrosine kinase gene: expression during embryonic angiogenesis.Oncogene 9, 395-403.

Korhonen, J., Lahtinen, I., Halmekyto, M., Alhonen, L., Janne, J., Dumont,D. and Alitalo, K. (1995). Endothelial-specific gene expression directed by thetie gene promoter in vivo. Blood 86, 1828-1835.

Kuhnert, F., Campagnolo, L., Xiong, J. W., Lemons, D., Fitch, M. J., Zou, Z.,Kiosses, W. B., Gardner, H. and Stuhlmann, H. (2005). Dosage-dependentrequirement for mouse Vezf1 in vascular system development. Dev. Biol. 283,140-156.

Lakso, M., Pichel, J. G., Gorman, J. R., Sauer, B., Okamoto, Y., Lee, E., Alt, F.W. and Westphal, H. (1996). Efficient in vivo manipulation of mouse genomicsequences at the zygote stage. Proc. Natl. Acad. Sci. USA 93, 5860-5865.

Makinen, T., Adams, R. H., Bailey, J., Lu, Q., Ziemiecki, A., Alitalo, K., Klein,R. and Wilkinson, G. A. (2005). PDZ interaction site in ephrinB2 is required forthe remodeling of lymphatic vasculature. Genes Dev. 19, 397-410.

Marron, M. B., Hughes, D. P., Edge, M. D., Forder, C. L. and Brindle, N. P.(2000). Evidence for heterotypic interaction between the receptor tyrosinekinases Tie-1 and Tie-2. J. Biol. Chem. 275, 39741-39746.

McCarthy, M. J., Burrows, R., Bell, S. C., Christie, G., Bell, P. R. F. and Brindle,N. P. (1999). Potential roles of metalloprotease mediated ectodomain cleavagein signaling by the endothelial receptor tyrosine kinase Tie-1. Lab. Invest. 79,889-895.

Morisada, T., Oike, Y., Yamada, Y., Urano, T., Akao, M., Kubota, Y.,Maekawa, H., Kimura, Y., Ohmura, M., Miyamoto, T. et al. (2005).Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation. Blood 105,4649-4656.

Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. and Roder, J. C.(1993). Derivation of completely cell culture-derived mice from early-passageembryonic stem cells. Proc. Natl. Acad. Sci. USA 90, 8424-8428.

Oliver, G. (2004). Lymphatic vasculature development. Nat. Rev. Immunol. 4, 35-45.

Oliver, G. and Detmar, M. (2002). The rediscovery of the lymphatic system: oldand new insights into the development and biological function of the lymphaticvasculature. Genes Dev. 16, 773-783.

Oliver, G. and Alitalo, K. (2005). The lymphatic vasculature: recent progress andparadigms. Annu. Rev. Cell Biol. 21, 457-483.

Oliver, G. and Srinivasan, R. S. (2008). Lymphatic vasculature development:current concepts. Ann. New York Acad. Sci. 1131, 75-81.

Partanen, J., Armstrong, E., Makela, T. P., Korhonen, J., Sandberg, M.,Renkonen, R., Knuutila, S., Huebner, K. and Alitalo, K. (1992). A novelendothelial cell surface receptor tyrosine kinase with extracellular epidermalgrowth factor homology domains. Mol. Cell. Biol. 12, 1698-1707.

Peters, K. G., Kontos, C. D., Lin, P. C., Wong, A. L., Rao, P., Huang, L.,Dewhirst, M. W. and Sankar, S. (2004). Functional significance of Tie2signaling in the adult vasculature. Recent Prog. Horm. Res. 59, 51-71.

Petrova, T. V., Karpanen, T., Norrmen, C., Mellor, R., Tamakoshi, T., Finegold,D., Ferrell, R., Kerjaschki, D., Mortimer, P., Yla-Herttuala, S. et al. (2004).Defective valves and abnormal mural cell recruitment underlie lymphatic vascularfailure in lymphedema distichiasis. Nat. Med. 10, 974-981.

Puri, M. C., Rossant, J., Alitalo, K., Bernstein, A. and Partanen, J. (1995). Thereceptor tyrosine kinase TIE is required for integrity and survival of vascularendothelial cells. EMBO J. 14, 5884-5891.

Puri, M. C., Partanen, J., Rossant, J. and Bernstein, A. (1999). Interaction ofthe TEK and TIE receptor tyrosine kinases during cardiovascular development.Development 126, 4569-4580.

Sabin, F. R. (1909). The lymphatic system in human embryos, with a considerationof the morphology of the system as a whole. Am. J. Anat. 9, 43-91.

Saharinen, P., Kerkelä, K., Ekman, N., Marron, M., Brindle, N., Lee, G. M.,Augustin, H., Koh, G. Y. and Alitalo, K. (2005). Multiple angiopoietinrecombinant proteins activate the Tie1 receptor tyrosine kinase and promote itsinteraction with Tie2. J. Cell Biol. 169, 239-243.

Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y.,Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W. and Qin, Y.

RESEARCH ARTICLE Development 137 (8)

DEVELO

PMENT

(1995). Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in bloodvessel formation. Nature 376, 70-74.

Schacht, V., Ramirez, M. I., Hong, Y. K., Hirakawa, S., Feng, D., Harvey, N.,Williams, M., Dvorak, A. M., Dvorak, H. F., Oliver, G. et al. (2003).T1a/podoplanin deficiency disrupts normal lymphatic vasculature formation andcauses lymphedema. EMBO J. 22, 3546-3556.

Singh, H., Milner, C. S., Aguilar Hernandez, M. M., Patel, N. and Brindle, N.P. (2009). Vascular endothelial growth factor activates the Tie family of receptortyrosine kinases. Cell. Signal. 21, 1346-1350.

Srinivasan, R. S., Dillard, M. E., Lagutin, O. V., Lin, F. J., Tsai, S., Tsai, M. J.,Samokhvalov, I. M. and Oliver, G. (2007). Lineage tracing demonstrates thevenous origin of the mammalian lymphatic vasculature. Genes Dev. 21, 2422-2432.

Taichman, D. B., Schachtner, S. K., Li, Y., Puri, M. C., Bernstein, A. andBaldwin, H. S. (2003). A unique pattern of Tie1 expression in the developingmurine lung. Exp. Lung Res. 29, 113-122.

Tammela, T., Saaristo, A., Lohela, M., Morisada, T., Tornberg, J., Norrmen,C., Oike, Y., Pajusola, K., Thurston, G., Suda, T. et al. (2005). Angiopoietin-1promotes lymphatic sprouting and hyperplasia. Blood 105, 4642-4648.

Wigle, J. T. and Oliver, G. (1999). Prox1 function is required for the developmentof the murine lymphatic system. Cell 98, 769-778.

Yabkowitz, R., Meyer, S., Black, T., Elliott, G., Merewether, L. A. andYamane, H. K. (1999). Inflammatory cytokines and vascular endothelial growthfactor stimulate the release of soluble Tie receptor from human endothelial cellsvia metalloprotease activation. Blood 93, 1969-1979.

Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J. andHolash, J. (2000). Vascular-specific growth factors and blood vessel formation.Nature 407, 242-248.

Yano, M., Iwama, A., Nishio, H., Suda, J., Takada, G. and Suda, T. (1997).Expression and function of murine receptor tyrosine kinases, TIE and TEK, inhematopoietic stem cells. Blood 89, 4317-4326.

Yuan, H. T., Venkatesha, S., Chan, B., Deutsch, U., Mammoto, T., Sukhatme,V. P., Woolf, A. S. and Karumanchi, S. A. (2007). Activation of the orphanendothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cellsurvival. FASEB J. 21, 3171-3183.

Yuan, L., Moyon, D., Pardanaud, L., Breant, C., Karkkainen, M. J., Alitalo, K.and Eichmann, A. (2002). Abnormal lymphatic vessel development inneuropilin 2 mutant mice. Development 129, 4797-4806.

1295RESEARCH ARTICLETie1 and lymphatic development

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