Abnormal Embryonic Lymphatic Vessel Development in Tie1 Hypomorphic Mice Xianghu Qu, Kevin Tompkins, Lorene E

CORRECTION

Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice Xianghu 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.

1417 RESEARCH ARTICLE 1285

Development 137, 1285-1295 (2010) doi:10.1242/dev.043380 © 2010. Published by The Company of Biologists Ltd Abnormal embryonic lymphatic vessel development in Tie1 hypomorphic mice Xianghu Qu1, Kevin Tompkins1, Lorene E. Batts1, Mira Puri2 and Scott Baldwin1,3,*

SUMMARY Tie1 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 is expressed at the earliest stages of lymphangiogenesis in Prox1-positive venous lymphatic endothelial cell (LEC) progenitors. LEC Tie1 expression is maintained throughout embryonic development and persists in postnatal mice. We then generated two lines of Tie1 mutant mice: a hypomorphic allele, which has reduced expression of Tie1, and a conditional allele. Reduction of Tie1 levels resulted in abnormal lymphatic patterning and in dilated and disorganized lymphatic vessels in all tissues examined and in impaired lymphatic drainage in embryonic skin. Homozygous hypomorphic mice also exhibited abnormally dilated jugular lymphatic vessels due to increased production of Prox1-positive LECs during initial lymphangiogenesis, indicating that Tie1 is required for the early stages of normal lymphangiogenesis. During later stages of lymphatic development, we observed an increase in LEC apoptosis in the 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 phenotypes observed correlated with the expression levels of Tie1, confirming a dosage dependence for Tie1 in LEC integrity and survival. No defects were observed in the arterial or venous vasculature. These results suggest that the developing lymphatic vasculature is particularly sensitive to alterations in Tie1 expression.

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

INTRODUCTION vein. These differentiating lymphatic endothelial cells (LECs) The lymphatic vascular system is a blind-ended network of sprout, migrate and proliferate to form primary lymph sacs in the endothelial cell-lined vessels essential for the maintenance of tissue jugular region (Oliver, 2004). Subsequently, several lymph sacs are fluid balance, immune surveillance and absorption of fatty acids in formed close to major veins in different regions of the embryo. The the gut. The lymphatic vessels are also involved in the pathogenesis primary lymphatic vascular plexus undergoes remodeling and of diseases such as tumor metastasis, lymphedema and various maturation to create a mature lymphatic network composed of large inflammatory conditions. Despite central roles in both normal and lymph vessels as well as an extensive lymphatic capillary network. disease physiology, our understanding of the development and Numerous signaling proteins have been identified as important in molecular regulation of the lymphatic vasculature lags far behind these later stages of the lymphatic development including ephrin B2 that of the parallel blood vascular system (Oliver, 2004; Oliver and (Makinen et al., 2005), neuropilin 2 (Yuan et al., 2002), angiopoietin Alitalo, 2005). 2 (Gale et al., 2002; Dellinger et al., 2008), podoplanin (Schacht et Many details about the development of the lymphatic vasculature al., 2003), integrin alpha 9 (Huang et al., 2000), and the transcription have been described only within the past decade, largely as a result factors Foxc2 (Petrova et al., 2004), Net (Ayadi et al., 2001), Vezf1 of studies of gene-targeted mice (Oliver and Srinivasan, 2008). For (Kuhnert et al., 2005), adrenomedullin (Fritz-Six et al., 2008) and mammals, the development of the lymphatic vessels in embryos is Aspp1 (Hirashima et al., 2008). initiated when a subset of endothelial cells in the cardinal vein sprout The orphan receptor tyrosine kinase (RTK) Tie1 shares a high to form the primary lymph sacs (Oliver and Detmar, 2002), degree of homology and is able to form heterodimers with Tie2, the confirming speculation of a venous origin made over a century ago receptor for the angiopoietins (Yancopoulos et al., 2000; Peters et (Sabin, 1909). In mice, the initiation of lymphatic differentiation al., 2004), and is known to play a major role in vascular is first discernible at embryonic day 10.5 (E10.5), when a development. Genetic studies in mice have demonstrated that Tie1 subpopulation of endothelial cells (ECs) expressing Lyve1, Prox1 is required for development and maintenance of the vascular system (Wigle and Oliver, 1999), Sox 18 (Francois et al., 2008; Hosking et as mice lacking Tie1 die in mid-gestation of hemorrhage and al., 2009) and Vegfr3 are detected on one side of the anterior cardinal defective microvessel integrity (Puri et al., 1995; Sato et al., 1995). Expression of Tie1 is restricted to ECs and to some hematopoietic cell lineages (Partanen et al., 1992; Korhonen et al., 1994; Dumont 1Division of Pediatric Cardiology, Vanderbilt University Medical Center, Nashville, et al., 1995; Hashiyama et al., 1996; Taichman et al., 2003; Yano et 2 TN 37232, USA. Sunnybrook and Women’s College Health Sciences Center, al., 1997). Interestingly, there is evidence that Tie1 is also expressed University of Toronto, Toronto, Ontario M4N 3M5, Canada. 3Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, by the initial and collecting lymphatic vessels in adult mice (Iljin et USA. al., 2002) and the first visible defect in the Tie1 mutants is edema (Sato et al., 1995). These observations led us to hypothesize that *Author for correspondence ([email protected]) Tie1 might serve a unique function in the development or

Accepted 5 February 2010 maintenance of the lymphatic vasculature. DEVELOPMENT 1286 RESEARCH ARTICLE Development 137 (8)

In this study, we found that Tie1 was expressed in the Prox1- mouse Vegfr3 antibody or rabbit anti-Prox1 and Alexa Fluor 594-conjugated positive venous LEC progenitors and lymphatic vessels donkey anti-goat IgG (Molecular Probes, A-11058) or Alexa Fluor 555 goat throughout embryonic and postnatal life. To circumvent early anti-rabbit IgG (Molecular Probes, A-21428). Lymphatic endothelial cell embryonic lethality observed in homozygous mutant animals, we (EC) proliferation was also determined by labeling with Prox1 and Ki67 generated mice with a conditional Tie1 allele that fortuitously (BD Biosciences, 550609). Other primary antibodies used in tissue section resulted in hypomorphic expression of Tie1. We were able to take immunohistochemistry were rat anti-mouse CD34 (eBioscience, 14-0341), advantage of this Tie1 hypomorphic allele to demonstrate a unique rat anti-mouse Icam1 (eBioscience, 14-0542), rat anti-mouse endoglin role for Tie1 in lymphatic development that was not observed in (eBioscience, 14-1051) and rat anti-mouse VE-cadherin (BD Biosciences, 555289). The percentage of proliferative lymphatic ECs in the cardinal vein the arterial or venous vasculature. Furthermore, the severity of the and jugular lymph sac areas of embryos at E11.5 to E13.5 was defined as the phenotypes observed correlated with the expression level of Tie1. number of BrdU+/Prox1+ cells divided by the total number of Prox1+ cells Our studies show that Tie1 is required for the early stages of in each field at similar level. normal lymphangiogenesis and is also involved in the later Apoptosis in lymphatic ECs was assed by TUNEL assay using ApopTag remodeling and stabilization of lymphatic vessels in a dosage- Plus Fluorescein in situ Cell Death Detection Kit (CHEMICON dependent manner. International, S7111). In addition, cleaved caspase 3 (Cell Signaling Technology, 9661) and Vegfr3 double staining was used to detect apoptotic MATERIALS AND METHODS LECs on frozen sections. Images were acquired on an Olympus fluorescent Generation of Tie1 mutant alleles microscope and processed in Adobe Photoshop. Percent apoptotic cells was loxP/loxP To generate Tie1 mice, a Tie1 floxed targeting vector was constructed determined by blinded quantification of TUNEL-positive cells in the based on the 129-Sv mouse genomic fragment used by Puri et al. (Puri et al., lymphatic or vascular endothelium divided by total Prox1 or DAPI-stained 1995). A 940 bp HpaI-SacI fragment containing a Tie1 minimal promoter nuclei in each comparable field. (Korhonen et al., 1995; Iljin et al., 2002) and exon 1 (containing the initial ATG codon) was inserted into the floxed KpnI-ClaI sites of the pDELBOY In situ hybridization plasmid (pDELBOY-3X), which contains an Frt-site-flanked neomycin In situ hybridization was performed on paraffin sections of 6 mm with gene (see Fig. S1 in the supplementary material). The targeting vector was radiolabeled single-stranded RNA probes. Probes were 0.42 kb mouse Tie1 electroporated into 129 R1 embryonic stem (ES) cells (Nagy et al., 1993) fragments corresponding to nucleotides 2221-2639 of NM_011587.2 and targeting was confirmed by Southern blotting with both 5Ј and 3Ј obtained by RT-PCR on mouse E18.5 lung RNA. external probes and PCR with the primers 5Ј-ATGCCTGTTCT AT - TTATTTTTCCAG-3Ј and 5Ј-TCGGGCGCGTTCAGAGTGGTAT-3Ј. Quantitative RT-PCR, northern analysis and western blotting Correctly targeted cells were then injected into C57/BL6 blastocysts and two RNA from the lungs of wild-type littermates, Tie1+/neo and Tie1neo/neo separate clones were found to transmit the targeted allele through the embryos at E18.5 was isolated using TRIzol Reagent (Invitrogen) with germline. Both lines were maintained on a 129-Sv and C57/BL6 background additional DNase treatment (Promega). cDNA was then generated using the and demonstrated identical phenotypes. SuperScript II Reverse Transcriptase Kit (Invitrogen). Quantitative PCR was performed on the LightCycler machine (Roche) using the LightCycler DNA Wholemount immunohistochemistry Master SYBR Green I Kit (Roche) and rodent Gapdh control reagents Hearts, diaphragm, limbs and head skin were collected from embryos and (Applied Biosystems). Sequences for the mouse Tie1 primers have been processed as previously described (Gale et al., 2002). The following primary published (Taichman et al., 2003). All assays were repeated at least twice antibodies were used: rat anti-mouse Pecam1 (Pharmingen, monoclonal and all samples were run in triplicate. MEC13.3), goat anti-mouse Vegfr3 antiserum (R&D Systems, #AF743), For northern analysis, RNA was hybridized with a probe based on the rabbit anti-Lyve1 (Upstate, #07-538) and Cy3-conjugated anti-a-smooth same 0.42 kb mouse Tie1 fragment used for in situ hybridization following muscle actin (SMA; Sigma, C-6198). gel electrophoresis and blots were stripped and rehybridized with a mouse For fluorescence immunostaining, the following secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-rabbit (Molecular Probes, glyceraldehyde-3-phosphate dehydrogenase (G3pdh) cDNA probe. A-11008), Cy3-conjugated donkey anti-goat and Cy2-conjugated donkey Western blotting was performed using standard protocols from anti-rat IgG (Jackson ImmunoResearch Laboratories, #705-165-147 and homogenized E18.5 lungs using primary antibody (rabbit anti-Tie1, Santa #712-225-150). Tissues were mounted in Vectashield (Vector Laboratories) Cruz Biotechnology) detected with secondary antibody (Alexa Fluor 488- and analyzed using a fluorescent microscope (Olympus) while images were conjugated goat anti-rabbit, Molecular Probes) and imaged with the Odyssey captured using a Zeiss LSM 510 confocal microscope. infrared scanner. Mouse b-actin was used as a control. To visualize anti-Lyve1 staining with light microscopy, biotinylated goat Lymphangiography anti-rabbit IgG (Vector Laboratories, BA-1000) and biotinylated rabbit anti- To visualize functional lymphatic vessels, FITC-dextran (Sigma, MW=2000 goat IgG (Vector Laboratories, BA-5000) secondary antibodies were used kDa, 8 mg/ml in PBS) was injected intradermally into the back of the in horseradish peroxidase stainings with the Vectastain Kit (Vector Elite PK- embryonic forelimb at E17.5 and E18.5 as previously described (Hirashima 6100) and DAB Kit (Vector Laboratories, SK-4100). et al., 2008). Lymphatic flow carrying FITC-dextran in embryos was X-gal staining, BrdU incorporation, TUNEL staining and section analyzed by Olympus fluorescence microscopy. For quantitative analysis of immunohistochemistry lymphangiography, the total length of lymphatic vessels carrying dye in X-gal staining was performed on frozen sections of tissues fixed in 4% embryos at E17.5 and E18.5 was measured using ImageJ software based on PFA/PBS from Tie1+/lacZ embryos as previously described (Puri et al., 1995) the photos taken after 2 minutes of injection. and tissues were then immunostained for lymphatics with primary antibody goat anti-mouse Vegfr3 and fluorescently labeled secondary antibody Cy3- Morphometric analysis conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories, The relative lymph sac size was quantified as described previously (Fritz- #705-165-147). Six et al., 2008) using both Lyve1-stained and Hematoxylin and Eosin BrdU incorporation was assessed in embryos following intraperitoneal (H&E)-stained paraffin sections. Briefly, transverse sections of the jugular injection of pregnant females with BrdU. For immunohistochemistry, mouse region of mutant and wild-type embryos were imaged on an Olympus anti-BrdU monoclonal antibody (clone G3G4, Developmental Studies SZX16 dissecting microscope. Sections containing jugular lymph sacs that Hybridoma Bank, University of Iowa, USA) and Alexa Fluor Cy2- were matched for the same anteroposterior level were blindly measured conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, #715-225- using ImageJ software. In order to normalize for section variability, the area

151) were used. Simultaneously, slides were also incubated with goat anti- of the lymph sac was divided by the area of the adjacent jugular vein. DEVELOPMENT Tie1 and lymphatic development RESEARCH ARTICLE 1287

Quantitative analysis of dermal lymphatic parameters (vessel diameter, vessel density and total vessel area per field) of wild-type, Tie1neo/neo and Tie1–/– embryos at E13.5 to E17.5 was performed as described (François et al., 2008), based on Vegfr3/Lyve1 wholemount immunostaining. Data are expressed as the means ± standard deviation. Statistical analysis was conducted using the two-tailed Student’s t-test. A P-value <0.05 was considered significant.

RESULTS Tie1 is expressed in the lymphatic vasculature throughout embryonic development We examined Tie1 expression at E10.5-E11.5 by X-gal staining of Tie1+/lacZ mice (Puri et al., 1995) and immunostaining with Prox1 antibody. The Tie1+/lacZ mice carry a b-gal reporter cassette inserted into exon 1 of the Tie1 locus under the control of the Tie1 promoter. This insertion allowed us to use b-gal detection to precisely follow Tie1 expression throughout development. We detected that Tie1 is co-expressed with Prox1 in LECs at all stages examined. As seen in Fig. 1A-C, a few endothelial cells lining the wall of the anterior cardinal vein were Prox1-positive. Some endothelial cells budding from the anterior cardinal vein were also Prox1-positive, which were located toward the outer margin of the vein. These cells represented a subpopulation of endothelial cells – the LECs. Almost all of these Prox1-positive cells co-expressed Tie1 (X-gal positive). The rest of the X-gal-positive/Prox1-negative cells corresponded to ECs of the vascular system. Next, we determined whether Tie1 is expressed in LECs at later stages. As the first detectable lymphatic vessels (jugular lymph sacs) in the mouse embryo can be morphologically distinguished from the blood vessels at E13.5, we examined Tie1 expression at E13.5 by X-gal staining of Tie1+/lacZ mice. As seen in Fig. 1D-E, at E13.5, X-gal (Tie1) stains not only the arteries, veins and blood capillaries, but also lymphatics, as judged initially by their anatomical position: in the neck region, the major lymphatic vessels are formed in close proximity to the cardinal vein. To precisely determine whether these vessels were lymphatics, we performed immunolabeling with antibody against Vegfr3, which is largely restricted to LEC after mid-gestation (Kaipainen et al., 1995). We detected strong Vegfr3 expression in the jugular lymphatic vessels Fig. 1. Tie1 expression in the LECs during embryogenesis. (Fig. 1E). Arteries did not express Vegfr3 but were clearly X-gal- +/lacZ positive. Veins were X-gal-positive but Vegfr3-negative. Therefore, (A-C) Transverse frozen section of E11.5 Tie1 mouse embryos double-labeled with X-gal (blue staining in A and C) and Prox1 Tie1 is expressed in the lymphatic endothelium during antibody (red in B and C). The polarized lymphatic endothelial cells embryogenesis at E13.5. The expression of Tie1 in the lymphatic (ECs) lining the wall of the anterior cardinal vein (cv) and budding from vessels at E13.5 was further confirmed by in situ hybridization (Fig. the anterior cv were labeled by Prox1 and were also X-gal-positive, 1F). indicating the co-expression of Prox1 and Tie1. X-gal-positive/Prox1- To determine whether Tie1 expression continues in lymphatic negative cells corresponded to ECs of the vascular system including vessels throughout development, we examined skin, intestine, capillaries and arteries (a). (D,E) Transverse frozen section through the mesentery and lung (Fig. 1G-J) from E17.5 embryos. As observed jugular region of an E13.5 Tie1+/lacZ embryo double-labeled with X-gal at earlier stages, X-gal staining in ECs colocalized with antibodies (D, blue) and Vegfr3 antibody (E, red). The Tie1-expressing cells were against Vegfr3 or Lyve1 (data not shown). Thus, Tie1 is expressed confirmed to be jugular lymphatic sacs (jls) by Vegfr3 labeling. X-gal- in the Prox1-positive venous LEC progenitors and lymphatic positive/Vegfr3-negative cells corresponded to ECs of the non- lymphatic vascular system. (F) Transverse section through the neck of an endothelia throughout embryonic development. E13.5 wild-type embryo hybridized with a murine Tie1 antisense riboprobe. Tie1 mRNA (red signal) is expressed in all ECs. Tie1 Characterization of a hypomorphic allele (G-J) Transverse frozen sections of skin (G), intestine (H), mesentery (I) To circumvent the embryonic and perinatal lethality observed in and lung (J) from E17.5 Tie1+/lacZ embryos double-labeled with X-gal homozygous-null mutant mice (Puri et al., 1995; Sato et al., 1995), (blue) and Vegfr3 antibody (red). Tie1 is not only expressed in blood we created a conditional allele of mouse Tie1 gene (see Fig. S1 in vessels and capillaries (X-gal-positive only), but also co-expressed with the supplementary material). Once the neomycin selection cassette Vegfr3 in the lymphatics (X-gal- and Vegfr3-positive, white arrows). was removed, the levels of Tiel expression in Tie1loxP/loxP mice were indistinguishable from those of wild-type animals. When these mice deleted pups (Tie1–/–) died of hemorrhage and severe edema in utero were crossed with E2A-Cre transgenic mice, which express Cre or at birth, displaying the same phenotypes of the conventional

recombinase in the germ line (Lakso et al., 1996), homozygous- knockout mice (Sato et al., 1995). DEVELOPMENT 1288 RESEARCH ARTICLE Development 137 (8)

null mutant Tie1–/– embryos exhibited extensive hemorrhage from E16.5 (Fig. 2F) to E18.5 or embryonic demise (Fig. 2G). Rare hypomorphic 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 was different from that in wild-type embryos, we performed northern blot analysis using embryonic lungs, a site of robust Tie1 expression during 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 in homozygous mutant lungs. We measured Tie1 mRNA by quantitative PCR analysis of the same samples, which showed that the Tie1 mRNA expression in the E18.5 lungs of Tie1+/neo and Tie1neo/neo embryos was reduced by 50.4% and 79.4%, respectively (Fig. 2I). Consistently, western blot analysis showed that Tie1 protein levels in the E18.5 lungs of Tie1+/neo and Tie1neo/neo embryos were reduced by 43.8% and 76.0%, respectively (Fig. 2J).

Tie1 attenuation results in enlarged lymph sacs We next examined sequential stages of lymphatic development in wild-type, Tie1-hypomorphic and Tie1-null mutant embryos. Histology of Tie1neo/neo and Tie1–/– embryos confirmed the presence of extensive interstitial edema (Fig. 3C,E). Lymph sacs were identified in wild-type, Tie1neo/neo and Tie1–/– embryos at E13.5 by immunohistochemical recognition of the lymphatic-specific makers Lyve1 (Fig. 3B,D,F) and Vegfr3 (data not shown), indicating normal lymphatic differentiation from venous vasculature. However, we observed that the jugular lymph sacs of Tie1neo/neo and Tie1–/– embryos appeared strikingly larger than those of litter-matched wild- Fig. 2. Characterization of the Tie1 hypomorphic allele. (A) A wild- type embryos. Computerized morphometry was used to calculate type embryo at E13.5. (B-D) Hypomorphic Tie1 homozygous embryos (neo/neo) demonstrating dorsal subcutaneous edema at E13.5 (B) and lymph sac area. At E13.5, the jugular lymphatic sacs in the wild-type E14.5 (D), and a Tie1 homozygous-null mutant embryo (–/–) at E13.5 embryos are located lateral to the internal jugular vein and dilate to (C) showing hemorrhage and dorsal edema. (E-G) A hypomorphic Tie1 a maximal diameter of approximately 2-3 times the size of the homozygous embryo displaying hemorrhage only at the tip of the tail diameter of the internal jugular vein (Fig. 3A,B) (Gittenberger-De and the tips of the toes at E16.5 (E) in contrast to the Tie1 Groot et al., 2004). In Tie1neo/neo and Tie1–/– embryos at E13.5, the homozygous-null mutant embryos which demonstrated severe maximal diameter of the jugular lymphatic sacs was approximately hemorrhage (F) or death (G) at E16.5. (H) Northern blot analysis of Tie1 10 times the size of the diameter of the internal jugular vein (Fig. +/neo mRNA expression level in the E18.5 lungs of wild-type, Tie1 and 3D,F,G). This phenotype persists until at least E15.5 (data not neo/neo Tie1 embryos. (I) Real-time RT-PCR showing that compared with shown). wild-type embryos (+/+), Tie1 mRNA expression levels in the E18.5 lungs of Tie1+/neo (+/neo) and Tie1neo/neo (neo/neo) embryos were reduced to 49.6% and 20.6%, respectively. (J) Western blot analysis of Tie1 attenuation leads to an early increase in Tie1 expression level in the E18.5 lungs of the three genotypes proliferation of LECs examined. To explore why the lymph sacs were dilated, we used a BrdU incorporation assay to assess proliferating cells in both lymph sacs and adjoining jugular veins. At E13.5, we observed no difference in Interestingly, when the neomycin selection cassette used in the the percentage of proliferative Prox1-positive ECs in lymphatic sacs initial targeting construct was not removed, the resultant Tie1neo (27.1±4.9% versus 25.6±4.1%) or jugular veins (data not shown) allele appeared to be hypomorphic. Tie1+/neo mice were bred to between the hypomorphic and wild-type embryos. However, when obtain homozygous pups, and mice homozygous for this allele embryos at E11.5 and at E12.5 were compared, the percentage of (Tie1neo/neo) displayed a more variable degree of severity than proliferative Prox1-positive cells in Tie1neo/neo embryos at E11.5 and detected in the null mutant animals. Before E16.5, the majority E12.5 was significantly higher than that in wild-type littermates (99/108, 91.7%) of the hypomorphic homozygous embryos (P<0.05), by 17.9% and 22.2%, respectively (Fig. 4). This increase exhibited mild to severe edema without signs of hemorrhage (Fig. of proliferative LECs was confirmed by co-labeling of adjacent 2B,D). By contrast, all of the Tie1–/– mutant embryos with the same sections with Prox1 and Ki67 (Fig. 4A,B). background manifested severe edema and 16.7% (4/24) also We then quantified the total number of LECs in the cardinal vein displayed localized hemorrhages distributed throughout the body and jugular lymph sac areas of embryos at sequential stages of surface. In Tie1–/– mutant embryos, the hemorrhagic defect was lymphatic development. At E10.5, the number of Prox1-positive usually observed from E13.5 onward (Fig. 2C), occasionally even LECs in Tie1neo/neo (Fig. 5B,J) and Tie1–/– (Fig. 5C,J) embryos were as early as E13.0 (data not shown). After E16.5, 34.6% (9/26) of increased by 23.2% and 29.1%, respectively (Fig. 5A). Similarly, hypomorphic embryos also developed hemorrhage but it was at E11.5, the number of LECs in Tie1neo/neo (Fig. 5E,J) and Tie1–/– localized to the tip of the tail, and occasionally (4/26) also at the tips (Fig. 5F,J) embryos were increased by 35.0% and 38.9%, neo/neo

of the toes (Fig. 2E). By contrast, the vast majority (92.5%) of the respectively, and at E12.5, the number of LECs in Tie1 (Fig. DEVELOPMENT Tie1 and lymphatic development RESEARCH ARTICLE 1289

Fig. 4. Tie1 attenuation during early embryonic development leads to an increase in LEC proliferation. (A-D) Transverse sections of wild-type littermates (A,C) and Tie1neo/neo (B,D) embryos through the jugular region at E11.5 (A,B) and E12.5 (C,D) were immunolabeled for Prox1 (red) and Ki67 (green) or Prox1 (red) and BrdU (green). Arrows Fig. 3. Tie1 attenuation leads to dilation of jugular lymph sacs at indicate representative proliferative Prox1-positive lymphatic endothelial E13.5. (A-F) Transverse sections through the jugular region of wild-type nuclei in the wall of the cardinal vein and jugular lymph sac areas. cv, littermates (A,B), Tie1neo/neo (C,D) and Tie1–/– (E,F) embryos at E13.5, cardinal vein; ls, lymph sac. Scale bar: 100 mm. (E) The percentage of labeled with Lyve1. (B,D,F) Higher magnification of the jugular region proliferative Prox1-positive cells in Tie1neo/neo embryos (gray bars) was (black triangle) in A, C and E, respectively. Note the remarkable increase significantly higher than that in wild-type littermates at E11.5 and at in size of lymph sacs relative to the jugular vein and carotid artery in the E12.5 (*, P<0.05), but no difference was seen at E13.5. Results hypomorphic (D) and mutant (F) embryos and a thickened dermis and represent the means ± the s.d. of 5 embryos per group. subcutaneous layer (double-headed arrows) in the hypomorphic (C) and mutant (E) embryonic skin when compared with the wild-type littermates (A). ( ) Quantification of lymph sac area, normalized to G Tie1 attenuation leads to an increase in lymphatic jugular vein area in hypomorphic embryos, mutant embryos and their wild-type littermates at E13.5. n>6 embryos per genotype. *, P<0.05. endothelial cell apoptosis jls, jugular lymph sacs; jv, jugular vein. Scale bars: 200 mm. To determine whether the abnormal lymphatic patterning of Tie1neo/neo mice could also be associated with abnormal EC apoptosis, we used TUNEL detection in conjunction with Vegfr3 labeling and DAPI nuclear localization to identify apoptosis. We 5H,J) and Tie1–/– (Fig. 5I,J) embryos were increased by 44.2% and detected no significant difference in the percentage of apoptotic 53.5%, respectively. These results suggest that an increased LECs in the wall of the cardinal vein and jugular lymph sac areas production of LECs at the initiation of lymphangiogenesis between wild-type (E11.5, 3.6±0.5%; E12.5, 3.2±0.8%) and contributes to the dilated lymph sacs seen in the Tie1 hypomorphic Tie1neo/neo (E11.5, 3.4±0.9%; E12.5, 2.9±1.1%) embryos (see Fig. and mutant mice. S3 in the supplementary material). However, at E13.5, we To evaluate whether Tie1 attenuation resulted in a global defect identified an increase in apoptosis in LECs of hypomorphic in endothelial differentiation, we used dual immunoflourescence at embryos. Only 2.5±0.6% TUNEL-positive cells were detected in E11.5 (Fig. 5D-F) and E12.5 (see Fig. S2 in the supplementary the lymphatic endothelium of the wild-type jugular lymphatic sacs material) with specific vascular EC markers – Pecam1, CD34, (Fig. 6A-C,G), whereas we observed 5.7±0.9% TUNEL-positive Icam1, endoglin or VE-cadherin and Prox1. We observed no LECs in the corresponding area of hypomorphic embryos (Fig. difference among wild-type, Tie1neo/neo and Tie1–/– embryos in the 6D-F,G). Interestingly, almost no EC apoptosis was seen in the expression of these five markers in vascular endothelium or at the jugular vein or arteries of wild-type or hypomorphic embryos. dorsal part of the cardinal vein, the site where lymphatic EC Taken together, these results demonstrate that reduction of Tie1 differentiation occurs during embryogenesis. Thus, the abnormal during embryonic development leads to a specific increase in dilated lymph sac defects in Tie1neo/neo and Tie1–/– embryos could lymphatic, but not venous, EC apoptosis at later stages of

not be ascribed to a primary endothelial defect. lymphangeogenesis. DEVELOPMENT 1290 RESEARCH ARTICLE Development 137 (8)

Fig. 6. Tie1 attenuation during embryonic development leads to 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 immunolabeled for Vegfr3 (red) and TUNEL (green). (G) The percentage of Vegfr3/TUNEL-positive apoptotic cells of the jugular lymph sacs was significantly higher in Tie1neo/neo embryos compared with wild-type littermates (*, P<0.05). Arrows indicate apoptotic LECs. cv, cardinal vein; jls, jugular lymph sac. Scale bar: 100 mm.

lymphatic capillaries (Fig. 7D) was detected in the head dermis near the developing ear and in limb skin. In the Tie1neo/neo embryos, however, the lymphatic capillaries in the corresponding regions were dilated and disorganized (Fig. 7B,E). The lymphatic defects in Fig. 5. Tie1 attenuation leads to an increased lymphatic the null mutant embryos (Fig. 7C,F) were more severe compared endothelial 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). with those in the hypomorphic embryos. Importantly, wholemount E12.5 sections (G-I) were labeled with Prox1 (red) and Vegfr3 (green). staining of E13.5 forelimb skin for Pecam1 revealed a relatively Representative images show more Prox1-positive LECs (arrows in A-C) normal blood vessel pattern in the hypomorphic embryos and only in the cardinal vein and jugular lymph sac areas in Tie1neo/neo (B,E,H) mildly dilated blood vessels in the null embryos (Fig. 7G-I), and Tie1–/– (C,F,I) embryos than in wild-type embryos (A,D,G). indicating a preferential effect on lymphatic vessels. These results Compared with wild-type control, both Tie1neo/neo and Tie1–/– embryos suggested that the severity of lymphatic defects in Tie1 mutant exhibit dilated jugular lymph sacs at E11.5 and at E12.5. cv, cardinal embryos is dependent on the dosage of Tie1 and that the vein; da, dorsal aorta; ls, lymph sac. Scale bars: 100 mm. Note: the development of lymphatic vasculature is more sensitive to Tie1 lymphatic marker Vegfr3 has weak expression in blood vessels at E12.5. reduction than the development of blood vasculature. (J) Quantitative analysis of the number of Prox1-positive nuclei in the To further investigate network formation of blood vessels and cardinal vein and jugular lymph sac areas of embryos at E10.5 to E12.5. *, P<0.05. lymphatic vessels, we performed wholemount double-fluorescence confocal microscopy of embryonic dorsal skin at later stages of development. As seen in Fig. S4 in the supplementary material, at Abnormal dermal lymphatic pattern in Tie1 E14.5, the Vegfr3-positive lymphatic capillary network was detected mutant embryos in wild-type control embryos, whereas lymphatic vessels were We next performed wholemount immunohistochemistry to examine disorganized and mildly dilated in Tie1neo/neo embryos; at E15.5, the development of dermal lymphatic vasculature in Tie1neo/neo lymphatic vessels in Tie1neo/neo embryos were dilated and lymphatic embryos at E13.5. In wild-type embryos, a few, but clear, Vegfr3- vessels started to regress in some areas, and compared with the neo/neo –/–

positive lymphatic vessels (Fig. 7A) and an extensive network of phenotype in Tie1 embryos, the lymphatic defects in Tie1 DEVELOPMENT Tie1 and lymphatic development RESEARCH ARTICLE 1291

diameter in the hypomorphic and mutant embryos at all developmental time points examined. The dilated dermal lymphatics seen in the Tie1 hypomorphic and mutant mice could be ascribed to the 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 was higher in Tie1neo/neo embryos than in wild-type embryos. This increase in LEC number was associated with an increase in lymphatic vessel area such that total LEC density (Prox1-positive nuclei/lymphatic vascular area) was not significantly altered in mutant 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 the hypomorphic and mutant embryos was significantly higher owing to vessel dilation at E13.5, but started to decrease compared with wild-type controls from E15.5 owing to vessel disruption or regression. Similar to the jugular lymphatic sacs, we also detected accentuated Vegfr3-positive EC apoptosis in the dermis of the hypomorphic embryos at E15.5 and at E17.5 with both TUNEL detection and antibodies against cleaved caspase 3, with minimal Vegfr3-positive EC apoptosis detected in wild-type embryos (see Fig. S8 in the supplementary material). Fig. 7. The severity of the dermal lymphatic defects in Tie1 By contrast, blood vascular patterning was relatively unaffected mutant embryos at E13.5 is dosage-dependent. (A-F) Heads (A-C) in the hypomorphic embryos at E14.5, E15.5 (data not shown), and forelimbs (D-F) of wild-type (A,D), Tie1neo/neo (B,E) and Tie1–/– (C,F) E16.5 (see Fig. S6A-C in the supplementary material) or E17.5 (Fig. embryos at E13.5 labeled with Vegfr3 antibody. Developing lymphatic 8C-L). Thus, attenuation in Tie1 was sufficient to alter the patterning network in the skin around the ear is normal in wild-type embryos (A), of the lymphatic vasculature without affecting the non-lymphatic but dilated and disorganized in Tie1neo/neo embryos (B), and even more –/– vasculature, suggesting that lymphatic dysfunction contributes to the disrupted in Tie1 embryos (C). Similarly, a normal network of edema seen in the Tie1 hypomorphic and mutant mice. This finding lymphatic capillaries was detected in the forelimb skin of wild-type embryos (D) and an irregular lymphatic network with dilated and also supports the hypothesis that developing lymphatics are more disorganized lymphatics was observed in the corresponding regions of sensitive to Tie1 reduction than the arterial or venous vasculature. neo/neo –/– Tie1 (E) and Tie1 (F) embryos, respectively. (G-I) Forelimbs of neo/neo wild-type (G), Tie1neo/neo (H) and Tie1–/– (I) embryos at E13.5 were Lymphatic function is impaired in Tie1 labeled with Pecam1 antibody and the blood vessel pattern appeared embryos normal. Arrowheads indicate the diameter of the lymphatic vessels. To assess whether structural defects seen in dermal lymphatic Scale bars: 250 mm. vessels lead to impaired lymphatic drainage, we evaluated lymphatic function by injecting high-molecular-weight FITC-dextran into embryonic limbs. Lymphangiography showed that dye uptake was embryos were even more severe. This was confirmed by Lyve1 rapid in the collecting lymphatic vessels and network of capillaries immunoreactivity of skin from the three genotypes at E15.5 (see Fig. in wild-type mice at E17.5 (Fig. 9A). However, the transport S5 in the supplementary material). function of lymphatic vessels in the hypomorphic embryos was By E16.5, the severity of dermal lymphatic defects in Tie1 impaired, consistent with the severity of the subcutaneous edema hypomorphic embryos was even more evident: Vegfr3-positive defect. As seen in Fig. 9B, FITC-dextran labeled only a few lymphatic vessels were dramatically dilated in some areas or disorganized lymphatic capillaries in the hypomorphic embryo that severely disrupted in other areas (see Fig. S6A-C in the had severe edema and localized hemorrhages at E17.5, indicating supplementary material). At E17.5 (Fig. 9; see Fig. S6D-F in the attenuation and/or poor function of the lymphatic capillary network. supplementary material), the Lyve1-positive lymphatic networks We obtained similar results using wild-type and hypomorphic were dense and well organized in wild-type embryos but were embryos at E18.5 (Fig. 9C). markedly dilated, disorganized and irregular in Tie1 hypomorphic embryo lymphatics. In addition, most of the wild-type lymphatic Lymphatic patterning is abnormal in internal capillaries were interconnected and only a few blind beginning organs of Tie1 hypomorphic embryos lymphatic capillaries were detected. By contrast, the formation of It is known that lymphatic capillaries progressively cover the heart lymphatic capillary networks was impaired in the head skin of the surface and the diaphragm from E15 onwards (Yuan et al., 2002). hypomorphic mice. The skin in the hypomorphic embryos exhibited Wholemount staining of these hearts with Lyve1 or Vegfr3 antibody incomplete network patterning with an increased number of blind revealed regression of the developing lymphatic vessels at the beginnings of lymphatics. Some regions were largely bereft of surface of the heart in the hypomorphic embryos compared with lymphatics (Fig. 8B,F; see Fig. S5 and Fig. S6 in the supplementary control littermates (see Fig. S9A-F in the supplementary material), material), indicating regression of the developing lymphatic vessels. which was first observed at E16.5. The epicardial lymphatic vessels To quantify the effect of Tie1 attenuation on lymphatic vessel in the hypomorphic embryos formed a primitive continuous network profiles, we measured lymphatic vessel area, density and diameter but were thinner than those in wild-type control embryos. In

(Fig. 8M-O). These studies documented an increase in vessel addition, the lymphatic vessels of the mutant embryos had begun to DEVELOPMENT 1292 RESEARCH ARTICLE Development 137 (8)

Fig. 8. Dermal lymphatic defects resulting 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 antibodies against Lyve1 (red) and Pecam1 (green) at E17.5. In comparison with control embryos, lymphatic networks in the skin of hypomorphic embryos were remarkably dilated, disorganized and regressed, but the non-lymphatic blood vessel pattern appeared normal. The arrow indicates regressing lymphatics. Arrowheads indicate the diameter of the lymphatic vessels. Original magnification of confocal images: ϫ10 (A-F); ϫ40 (G-L). Scale bars: 100 mm. (M-O) Quantitative analysis of lymphatic parameters in the skin of wild-type, Tie1neo/neo and Tie1–/– embryos at E13.5 to E17.5 based on Vegfr3/Lyve1 wholemount immunostaining. (M) Vessel size (diameter). (N) Vessel density. (O) Total vessel area per field. *, P<0.05.

regress in some areas. At E17.5, the entire lymphatic network was persisted in the postnatal animal. The early expression in the disrupted in the hypomorphic embryos and by E18.5, only a few lymphatic endothelium suggests a unique role for Tie1 in the single disrupted lymphatic vessels were detected on the surface of development and function of the lymphatic vessels. the heart. In the central portion of the diaphragm at E16.5, some of The intronic insertion of a neo cassette causes aberrant splicing of the larger collecting lymphatics in the hypomorphic embryos were Tie1 transcripts, reducing the amount of mRNA encoding functional dilated and disorganized compared with wild-type embryos (see Fig. Tie1 protein to ~20% of normal levels. This Tie1 hypomorphic allele S9G-L in the supplementary material). By E17.5 and E18.5, there enabled us to demonstrate that a dosage-dependent reduction of Tie1 was a striking reduction in the number of lymphatics in the whole results in progressive defects in lymphatic patterning not seen in other diaphragm of the hypomorphic embryos. Furthermore, all of the EC populations. Tie1 attenuation resulted in significantly enlarged lymphatics were much thinner than those in wild-type control jugular lymphatic vessels. Consistent with structural defects seen in embryos and the network was disrupted, indicating regression of the the lymphatics, lymphatic function was also impaired in hypomorphic developing lymphatics over the entire diaphragmatic surface. Taken embryonic skin. We detected no apparent lymphatic defects in the together, the Tie1neo/neo mice demonstrated abnormally patterned heterozygous Tie1+/neo embryos at any developmental stage. lymphatic vessels in all internal organs examined. Interestingly, although the Tie1 hypomorphic embryos display severe generalized edema, there was almost no apparent hemorrhage DISCUSSION during the entire period of embryonic development. A few embryos Genetic studies in mice have demonstrated that Tie1 is required for developed hemorrhage at the tips of the tail or digits and this development and maintenance of the vascular system. Deletion of occurred at a much later time point than the diffuse hemorrhage Tie1 leads to embryonic lethality due to edema, hemorrhage and observed in the complete null embryos. In addition, although microvessel rupture (Puri et al., 1995; Sato et al., 1995). lymphatic defects were obvious in most of the hypomorphic Interestingly, the timing of previously reported embryonic demise embryos, the pattern of blood vessels appeared relatively normal. In at E13.5 correlated with the earliest time when lymphatic vessels can fact, when we examined expression of the specific blood vascular be morphologically distinguished from the blood vessels. The EC markers in the major blood vessels and in particular at the dorsal previous report (Sato et al., 1995) and our study showed that the first part of the cardinal vein, the site where lymphatic EC differentiation visible defect in the Tie1 mutants is dorsal subcutaneous edema from occurs during the initiation of lymphangiogenesis, we could detect E13.5 onward. In this report, we have documented that Tie1 is no differences in the pattern of expression between wild-type and expressed in LEC progenitors at E10.5 and E11.5 before lymphatic hypomorphic mutant embryos. These results further suggest that the vessels are formed. This expression of Tie1 in the lymphatic development of lymphatic vasculature is more sensitive to Tie1

endothelium is maintained throughout embryonic development and reduction than the development of blood vasculature. DEVELOPMENT Tie1 and lymphatic development RESEARCH ARTICLE 1293

This early period of increased LEC proliferation was followed by an accentuation of apoptosis in the latter stages of lymphatic vascular development. We found normal LEC apoptosis in Tie1 hypomorphic and mutant embryos at early stages (E11.5 and E12.5) but a selective increase in LEC apoptosis from E13.5. This is consistent with the observed regression of the already-formed developing lymphatics and the previous report that Tie1 inhibits apoptosis and is required for EC survival (Kontos, et al., 2002). From these findings, we conclude that attenuation of Tie1 in lymphatics results in dilation due to an elevated production of LECs during specification of lymphatic vasculature, and regression after mid-gestation due to increased apoptosis of LECs. This process leads to a disorganized and disrupted lymphatic network. The lymphatic defects of Tie1 mutant mice are reminiscent of the lymphatic phenotypes of the Tie2 ligand, Ang2, mutant mice, although the latter exhibits only dilated lymphatic vessels without regression and with no defects in the size of jugular lymphatic sacs (Gale et al., 2002; Dellinger et al., 2008). Ang2 is involved in the remodeling and stabilization of lymphatic vessels. Ang2–/– mice exhibit defects in the remodeling of the blood vasculature, but surprisingly, even more severe defects of the lymphatic system. Ang2–/– mice display chylous ascites, peripheral lymphedema and hypoplasia of the lymphatic vasculature (Gale et al., 2002). Lymphatic vessels in Ang2–/– mice fail to mature and do not exhibit Fig. 9. Tie1 attenuation leads to impaired lymphatic drainage a collecting vessel phenotype. Furthermore, dermal lymphatic function. (A,B) Representative image of the subcutaneous collecting vessels in Ang2–/– pups prematurely recruit smooth muscle cells lymphatic vessels (arrows) and lymphatic capillaries (arrowheads) using and do not undergo proper postnatal remodeling (Dellinger et al., fluorescence microscopy after intradermal FITC-dextran injection into 2008). embryonic forelimbs at E17.5. Sites of injection are indicated by dashed To date, the Tie1 receptor remains primarily an orphan receptor, neo/neo circles. The lymphatic network in Tie1 embryos (B) is disorganized as no ligand for Tie1 has been identified and only ligand- and uptake of FITC-dextran is attenuated when compared with that independent signal transduction pathways have been characterized seen in wild-type mice (A). Scale bar: 500 mm. (C) Quantitative analysis of the total length of lymphatic vessels (2 minutes after each injection) (McCarthy et al., 1999; Yabkowitz et al., 1999). However, Tie1 in wild-type versus Tie1neo/neo embryos at E17.5 and at E18.5. phosphorylation can be induced by overexpression of multiple *, P<0.05. angiopoietin proteins (Ang1 and Ang4) in vitro and activation is amplified via association with Tie2 (Saharinen et al., 2005). Tie1 and Tie2 receptor heterodimers had been shown to exist in cultured ECs It is commonly thought that most of the lymphovascular system (Marron et al., 2000) but the cellular function of heterodimerization in mice is of venous origin (Oliver, 2004; Oliver and Alitalo, 2005). remains to be determined. As Tie1 is expressed in the endothelium However, recent data argues for a dual origin of LECs in the mouse of the same lymphatics as Ang2 during embryogenesis and with incorporation of mesenchymal lymphangioblast-derived cells enhanced Tie1 mRNA expression occurs together with Ang2 mRNA into the lining of lymph vessels during development providing a in the adults (Gale et al., 2002; Iljin et al., 2002), it is tempting to minor contribution to the lymphatics (Buttler et al., 2006; Buttler et speculate that Tie1 might be modulating Ang2 function via al., 2008). Although our work does not directly address whether all interaction with the Tie2 receptor in the lymphatic ECs as has been lymphatics are of venous origin or whether there is an additional documented in cultured ECs (Yuan et al., 2007) and endothelial contribution from a mesenchymal cell population, the fact that we progenitor cells (Kim et al., 2006). Interestingly, investigators have observed similar abnormalities in both the central lymphatics as well recently shown that Vegf can cause a 4-fold increase in as in the dermis of the embryo would suggest that Tie1 is required phosphorylation of Tie2 that is independent of angiopoietin for the normal development of both the central lymphatics and the expression but dependent on proteolytic cleavage of Tie1 and peripheral lymphatics. subsequent transphosphorylation of Tie2, further supporting the Jugular lymphatic sac dilation was a prominent feature in the Tie1 possibility of Tie1 modulation of Tie2 signaling (Singh et al., 2009). mutant embryos. Attenuation of Tie1 leads to an increased LEC However, the expression of Tie2 and its role in LEC development production before mid-gestation in the Tie1 hypomorphic and has not been well defined. Adult Lyve1-positive lymphatic mutant mice. Consistently, we detected significantly more vessels have previously been shown to express Tie2 by proliferative LECs in the cardinal vein and jugular lymph sac areas immunofluorescence labeling of mouse ear skin and the small of Tie1 hypomorphic and mutant embryos at E11.5 and E12.5, when intestine using an antibody against Tie2 (Morisada et al., 2005; specification of lymphatic vasculature occurs (Francois et al., 2008). Tammela et al., 2005). However, other groups failed to detect GFP Although the proliferation rate of LECs in jugular lymphatic sacs of expression by lymphatic vessels in the adult (Dellinger et al., 2008) Tie1 hypomorphic and mutant embryos at E13.5 returns to normal, or embryos (Srinivasan et al., 2007) from Tie2-GFP transgenic mice the absolute total number of proliferative LECs is still higher than using either immunohistochemical (GFP) analyses or in situ that in wild-type embryos because the absolute number of LECs is hybridization. One possible explanation for this discrepancy is that

higher (Fig. 5J). the regulatory elements controlling the expression of Tie2 by DEVELOPMENT 1294 RESEARCH ARTICLE Development 137 (8) lymphatic vessels are not included in the Tie2-GFP transgenic Iljin, K., Petrova, T. V., Veikkola, T., Kumar, V., Poutanen, M. and Alitalo, K. construct. Further delineation of the expression pattern of Tie2 (2002). A fluorescent Tie1 reporter allows monitoring of vascular development and endothelial cell isolation from transgenic mouse embryos. FASEB J. 16, should resolve this discrepancy. 1764-1774. In summary, it is now evident that the Tie receptors and their Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, V. W., Fang, G. ligands demonstrate context-dependent regulation of vascular H., Dumont, D., Breitman, M. and Alitalo, K. (1995). Expression of the fms- remodeling (Eklund et al., 2006) and our work suggests a unique like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. USA 92, 3566-3570. dosage-dependent role for Tie1 in lymphatic ontogeny. Whether Kim, K. L., Shin, I. S., Kim, J. M., Choi, J. H., Byun, J., Jeon, E. S., Suh, W. and Tie1 signals independently or in association with Tie2 is a focus of Kim, D. K. (2006). Interaction between Tie receptors modulates angiogenic ongoing investigation. However, the development of a activity of angiopoietin2 in endothelial progenitor cells. Cardiovasc. Res. 72, 394-402. hypomorphic, conditional Tie1 allele in combination with the Kontos, C. D., Cha, E. H., York, J. D. and Peters, K. G. (2002). The endothelial available lymphatic-specific Cre deletor lines (Srinivasan et al., receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to 2007) should provide the necessary reagents for the temporal- and inhibit apoptosis. Mol. Cell. Biol. 22, 1704-1713. lymphatic-specific deletion required for further investigation. Korhonen, J., Polvi, A., Partanen, J. and Alitalo, K. (1994). The mouse tie receptor tyrosine kinase gene: expression during embryonic angiogenesis. Oncogene 9, 395-403. Acknowledgements Korhonen, J., Lahtinen, I., Halmekyto, M., Alhonen, L., Janne, J., Dumont, We thank Dr Lawrence Price (Vanderbilt University) for providing the antibodies D. and Alitalo, K. (1995). Endothelial-specific gene expression directed by the against Prox1 and valuable discussions and Dr Christopher Brown (Vanderbilt tie gene promoter in vivo. Blood 86, 1828-1835. University) for critical reading of the manuscript. This work was supported by Kuhnert, F., Campagnolo, L., Xiong, J. W., Lemons, D., Fitch, M. J., Zou, Z., NIH grants R01 HL086964 (S.B.) and DK038517 and a grant-in-aid from the Kiosses, W. B., Gardner, H. and Stuhlmann, H. (2005). Dosage-dependent March of Dimes. Deposited in PMC for release after 12 months. requirement for mouse Vezf1 in vascular system development. Dev. Biol. 283, 140-156. Competing interests statement Lakso, M., Pichel, J. G., Gorman, J. 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5215. Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W. and Qin, Y. DEVELOPMENT Tie1 and lymphatic development RESEARCH ARTICLE 1295

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