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© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 1417 doi:10.1242/dev.108969
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.
INTRODUCTION
The lymphatic vascular system is a blind-ended network of
endothelial cell-lined vessels essential for the maintenance of tissue
fluid balance, immune surveillance and absorption of fatty acids in
the gut. The lymphatic vessels are also involved in the pathogenesis
of diseases such as tumor metastasis, lymphedema and various
inflammatory conditions. Despite central roles in both normal and
disease physiology, our understanding of the development and
molecular regulation of the lymphatic vasculature lags far behind
that of the parallel blood vascular system (Oliver, 2004; Oliver and
Alitalo, 2005).
Many details about the development of the lymphatic vasculature
have been described only within the past decade, largely as a result
of studies of gene-targeted mice (Oliver and Srinivasan, 2008). For
mammals, the development of the lymphatic vessels in embryos is
initiated when a subset of endothelial cells in the cardinal vein sprout
to 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 differentiation
is first discernible at embryonic day 10.5 (E10.5), when a
subpopulation of endothelial cells (ECs) expressing Lyve1, Prox1
(Wigle and Oliver, 1999), Sox 18 (Francois et al., 2008; Hosking et
al., 2009) and Vegfr3 are detected on one side of the anterior cardinal
1
Division 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 and
Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232,
USA.
*Author for correspondence ([email protected])
Accepted 5 February 2010
vein. These differentiating lymphatic endothelial cells (LECs)
sprout, migrate and proliferate to form primary lymph sacs in the
jugular region (Oliver, 2004). Subsequently, several lymph sacs are
formed close to major veins in different regions of the embryo. The
primary lymphatic vascular plexus undergoes remodeling and
maturation to create a mature lymphatic network composed of large
lymph vessels as well as an extensive lymphatic capillary network.
Numerous signaling proteins have been identified as important in
these later stages of the lymphatic development including ephrin B2
(Makinen et al., 2005), neuropilin 2 (Yuan et al., 2002), angiopoietin
2 (Gale et al., 2002; Dellinger et al., 2008), podoplanin (Schacht et
al., 2003), integrin alpha 9 (Huang et al., 2000), and the transcription
factors Foxc2 (Petrova et al., 2004), Net (Ayadi et al., 2001), Vezf1
(Kuhnert et al., 2005), adrenomedullin (Fritz-Six et al., 2008) and
Aspp1 (Hirashima et al., 2008).
The orphan receptor tyrosine kinase (RTK) Tie1 shares a high
degree of homology and is able to form heterodimers with Tie2, the
receptor for the angiopoietins (Yancopoulos et al., 2000; Peters et
al., 2004), and is known to play a major role in vascular
development. Genetic studies in mice have demonstrated that Tie1
is required for development and maintenance of the vascular system
as mice lacking Tie1 die in mid-gestation of hemorrhage and
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
et al., 1995; Hashiyama et al., 1996; Taichman et al., 2003; Yano et
al., 1997). Interestingly, there is evidence that Tie1 is also expressed
by the initial and collecting lymphatic vessels in adult mice (Iljin et
al., 2002) and the first visible defect in the Tie1 mutants is edema
(Sato et al., 1995). These observations led us to hypothesize that
Tie1 might serve a unique function in the development or
maintenance of the lymphatic vasculature.
DEVELOPMENT
KEY WORDS: Tie1, Lymphatic development, Hypomorphic, Proliferation, Apoptosis, Mouse
1286 RESEARCH ARTICLE
MATERIALS AND METHODS
Generation of Tie1 mutant alleles
To generate Tie1loxP/loxP mice, a Tie1 floxed targeting vector was constructed
based 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 initial
ATG codon) was inserted into the floxed KpnI-ClaI sites of the pDELBOY
plasmid (pDELBOY-3X), which contains an Frt-site-flanked neomycin
gene (see Fig. S1 in the supplementary material). The targeting vector was
electroporated 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⬘-ATGCCTGTTCTATTTATTTTTCCAG-3⬘ and 5⬘-TCGGGCGCGTTCAGAGTGGTAT-3⬘.
Correctly targeted cells were then injected into C57/BL6 blastocysts and two
separate clones were found to transmit the targeted allele through the
germline. Both lines were maintained on a 129-Sv and C57/BL6 background
and demonstrated identical phenotypes.
Wholemount immunohistochemistry
Hearts, diaphragm, limbs and head skin were collected from embryos and
processed as previously described (Gale et al., 2002). The following primary
antibodies were used: rat anti-mouse Pecam1 (Pharmingen, monoclonal
MEC13.3), goat anti-mouse Vegfr3 antiserum (R&D Systems, #AF743),
rabbit anti-Lyve1 (Upstate, #07-538) and Cy3-conjugated anti-a-smooth
muscle actin (SMA; Sigma, C-6198).
For fluorescence immunostaining, the following secondary antibodies
were used: Alexa Fluor 488-conjugated goat anti-rabbit (Molecular Probes,
A-11008), Cy3-conjugated donkey anti-goat and Cy2-conjugated donkey
anti-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 were
captured using a Zeiss LSM 510 confocal microscope.
To visualize anti-Lyve1 staining with light microscopy, biotinylated goat
anti-rabbit IgG (Vector Laboratories, BA-1000) and biotinylated rabbit antigoat IgG (Vector Laboratories, BA-5000) secondary antibodies were used
in horseradish peroxidase stainings with the Vectastain Kit (Vector Elite PK6100) and DAB Kit (Vector Laboratories, SK-4100).
X-gal staining, BrdU incorporation, TUNEL staining and section
immunohistochemistry
X-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 antibody
goat anti-mouse Vegfr3 and fluorescently labeled secondary antibody Cy3conjugated donkey anti-goat IgG (Jackson ImmunoResearch Laboratories,
#705-165-147).
BrdU incorporation was assessed in embryos following intraperitoneal
injection of pregnant females with BrdU. For immunohistochemistry, mouse
anti-BrdU monoclonal antibody (clone G3G4, Developmental Studies
Hybridoma Bank, University of Iowa, USA) and Alexa Fluor Cy2conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, #715-225151) were used. Simultaneously, slides were also incubated with goat anti-
mouse Vegfr3 antibody or rabbit anti-Prox1 and Alexa Fluor 594-conjugated
donkey anti-goat IgG (Molecular Probes, A-11058) or Alexa Fluor 555 goat
anti-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 section
immunohistochemistry 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 vein
and jugular lymph sac areas of embryos at E11.5 to E13.5 was defined as the
number of BrdU+/Prox1+ cells divided by the total number of Prox1+ cells
in each field at similar level.
Apoptosis in lymphatic ECs was assed by TUNEL assay using ApopTag
Plus Fluorescein in situ Cell Death Detection Kit (CHEMICON
International, S7111). In addition, cleaved caspase 3 (Cell Signaling
Technology, 9661) and Vegfr3 double staining was used to detect apoptotic
LECs on frozen sections. Images were acquired on an Olympus fluorescent
microscope and processed in Adobe Photoshop. Percent apoptotic cells was
determined by blinded quantification of TUNEL-positive cells in the
lymphatic or vascular endothelium divided by total Prox1 or DAPI-stained
nuclei in each comparable field.
In situ hybridization
In situ hybridization was performed on paraffin sections of 6 mm with
radiolabeled single-stranded RNA probes. Probes were 0.42 kb mouse Tie1
fragments corresponding to nucleotides 2221-2639 of NM_011587.2
obtained by RT-PCR on mouse E18.5 lung RNA.
Quantitative RT-PCR, northern analysis and western blotting
RNA from the lungs of wild-type littermates, Tie1+/neo and Tie1neo/neo
embryos at E18.5 was isolated using TRIzol Reagent (Invitrogen) with
additional DNase treatment (Promega). cDNA was then generated using the
SuperScript II Reverse Transcriptase Kit (Invitrogen). Quantitative PCR was
performed on the LightCycler machine (Roche) using the LightCycler DNA
Master SYBR Green I Kit (Roche) and rodent Gapdh control reagents
(Applied Biosystems). Sequences for the mouse Tie1 primers have been
published (Taichman et al., 2003). All assays were repeated at least twice
and all samples were run in triplicate.
For northern analysis, RNA was hybridized with a probe based on the
same 0.42 kb mouse Tie1 fragment used for in situ hybridization following
gel electrophoresis and blots were stripped and rehybridized with a mouse
glyceraldehyde-3-phosphate dehydrogenase (G3pdh) cDNA probe.
Western blotting was performed using standard protocols from
homogenized E18.5 lungs using primary antibody (rabbit anti-Tie1, Santa
Cruz Biotechnology) detected with secondary antibody (Alexa Fluor 488conjugated goat anti-rabbit, Molecular Probes) and imaged with the Odyssey
infrared scanner. Mouse b-actin was used as a control.
Lymphangiography
To visualize functional lymphatic vessels, FITC-dextran (Sigma, MW=2000
kDa, 8 mg/ml in PBS) was injected intradermally into the back of the
embryonic forelimb at E17.5 and E18.5 as previously described (Hirashima
et al., 2008). Lymphatic flow carrying FITC-dextran in embryos was
analyzed by Olympus fluorescence microscopy. For quantitative analysis of
lymphangiography, the total length of lymphatic vessels carrying dye in
embryos at E17.5 and E18.5 was measured using ImageJ software based on
the photos taken after 2 minutes of injection.
Morphometric analysis
The relative lymph sac size was quantified as described previously (FritzSix et al., 2008) using both Lyve1-stained and Hematoxylin and Eosin
(H&E)-stained paraffin sections. Briefly, transverse sections of the jugular
region of mutant and wild-type embryos were imaged on an Olympus
SZX16 dissecting microscope. Sections containing jugular lymph sacs that
were matched for the same anteroposterior level were blindly measured
using ImageJ software. In order to normalize for section variability, the area
of the lymph sac was divided by the area of the adjacent jugular vein.
DEVELOPMENT
In this study, we found that Tie1 was expressed in the Prox1positive venous LEC progenitors and lymphatic vessels
throughout embryonic and postnatal life. To circumvent early
embryonic lethality observed in homozygous mutant animals, we
generated mice with a conditional Tie1 allele that fortuitously
resulted in hypomorphic expression of Tie1. We were able to take
advantage of this Tie1 hypomorphic allele to demonstrate a unique
role for Tie1 in lymphatic development that was not observed in
the arterial or venous vasculature. Furthermore, the severity of the
phenotypes observed correlated with the expression level of Tie1.
Our studies show that Tie1 is required for the early stages of
normal lymphangiogenesis and is also involved in the later
remodeling and stabilization of lymphatic vessels in a dosagedependent manner.
Development 137 (8)
Tie1 and lymphatic development
RESEARCH ARTICLE 1287
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. 1E). Arteries did not express Vegfr3 but were clearly X-galpositive. Veins were X-gal-positive but Vegfr3-negative. Therefore,
Tie1 is expressed in the lymphatic endothelium during
embryogenesis at E13.5. The expression of Tie1 in the lymphatic
vessels at E13.5 was further confirmed by in situ hybridization (Fig.
1F).
To determine whether Tie1 expression continues in lymphatic
vessels throughout development, we examined skin, intestine,
mesentery and lung (Fig. 1G-J) from E17.5 embryos. As observed
at earlier stages, X-gal staining in ECs colocalized with antibodies
against Vegfr3 or Lyve1 (data not shown). Thus, Tie1 is expressed
in the Prox1-positive venous LEC progenitors and lymphatic
endothelia throughout embryonic development.
Characterization of a hypomorphic Tie1 allele
To circumvent the embryonic and perinatal lethality observed in
homozygous-null mutant mice (Puri et al., 1995; Sato et al., 1995),
we created a conditional allele of mouse Tie1 gene (see Fig. S1 in
the supplementary material). Once the neomycin selection cassette
was removed, the levels of Tiel expression in Tie1loxP/loxP mice were
indistinguishable from those of wild-type animals. When these mice
were crossed with E2A-Cre transgenic mice, which express Cre
recombinase in the germ line (Lakso et al., 1996), homozygous-
Fig. 1. Tie1 expression in the LECs during embryogenesis.
(A-C) Transverse frozen section of E11.5 Tie1+/lacZ mouse embryos
double-labeled with X-gal (blue staining in A and C) and Prox1
antibody (red in B and C). The polarized lymphatic endothelial cells
(ECs) lining the wall of the anterior cardinal vein (cv) and budding from
the anterior cv were labeled by Prox1 and were also X-gal-positive,
indicating the co-expression of Prox1 and Tie1. X-gal-positive/Prox1negative cells corresponded to ECs of the vascular system including
capillaries and arteries (a). (D,E) Transverse frozen section through the
jugular region of an E13.5 Tie1+/lacZ embryo double-labeled with X-gal
(D, blue) and Vegfr3 antibody (E, red). The Tie1-expressing cells were
confirmed to be jugular lymphatic sacs (jls) by Vegfr3 labeling. X-galpositive/Vegfr3-negative cells corresponded to ECs of the nonlymphatic vascular system. (F) Transverse section through the neck of an
E13.5 wild-type embryo hybridized with a murine Tie1 antisense
riboprobe. 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 blood
vessels and capillaries (X-gal-positive only), but also co-expressed with
Vegfr3 in the lymphatics (X-gal- and Vegfr3-positive, white arrows).
deleted pups (Tie1–/–) died of hemorrhage and severe edema in utero
or at birth, displaying the same phenotypes of the conventional
knockout mice (Sato et al., 1995).
DEVELOPMENT
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.
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 wildtype 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).
Interestingly, when the neomycin selection cassette used in the
initial targeting construct was not removed, the resultant Tie1neo
allele appeared to be hypomorphic. Tie1+/neo mice were bred to
obtain homozygous pups, and mice homozygous for this allele
(Tie1neo/neo) displayed a more variable degree of severity than
detected in the null mutant animals. Before E16.5, the majority
(99/108, 91.7%) of the hypomorphic homozygous embryos
exhibited mild to severe edema without signs of hemorrhage (Fig.
2B,D). By contrast, all of the Tie1–/– mutant embryos with the same
background manifested severe edema and 16.7% (4/24) also
displayed localized hemorrhages distributed throughout the body
surface. In Tie1–/– mutant embryos, the hemorrhagic defect was
usually observed from E13.5 onward (Fig. 2C), occasionally even
as early as E13.0 (data not shown). After E16.5, 34.6% (9/26) of
hypomorphic embryos also developed hemorrhage but it was
localized to the tip of the tail, and occasionally (4/26) also at the tips
of the toes (Fig. 2E). By contrast, the vast majority (92.5%) of the
Tie1 attenuation leads to an early increase in
proliferation of LECs
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
the 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, when
embryos at E11.5 and at E12.5 were compared, the percentage of
proliferative Prox1-positive cells in Tie1neo/neo embryos at E11.5 and
E12.5 was significantly higher than that in wild-type littermates
(P<0.05), by 17.9% and 22.2%, respectively (Fig. 4). This increase
of proliferative LECs was confirmed by co-labeling of adjacent
sections with Prox1 and Ki67 (Fig. 4A,B).
We then quantified the total number of LECs in the cardinal vein
and jugular lymph sac areas of embryos at sequential stages of
lymphatic development. At E10.5, the number of Prox1-positive
LECs in Tie1neo/neo (Fig. 5B,J) and Tie1–/– (Fig. 5C,J) embryos were
increased 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.
DEVELOPMENT
Fig. 2. Characterization of the Tie1 hypomorphic allele. (A) A wildtype embryo at E13.5. (B-D) Hypomorphic Tie1 homozygous embryos
(neo/neo) demonstrating dorsal subcutaneous edema at E13.5 (B) and
E14.5 (D), and a Tie1 homozygous-null mutant embryo (–/–) at E13.5
(C) showing hemorrhage and dorsal edema. (E-G) A hypomorphic Tie1
homozygous embryo displaying hemorrhage only at the tip of the tail
and the tips of the toes at E16.5 (E) in contrast to the Tie1
homozygous-null mutant embryos which demonstrated severe
hemorrhage (F) or death (G) at E16.5. (H) Northern blot analysis of Tie1
mRNA expression level in the E18.5 lungs of wild-type, Tie1+/neo and
Tie1neo/neo embryos. (I) Real-time RT-PCR showing that compared with
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 expression level in the E18.5 lungs of the three genotypes
examined.
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 wildtype embryos. Computerized morphometry was used to calculate
lymph sac area. At E13.5, the jugular lymphatic sacs in the wild-type
embryos are located lateral to the internal jugular vein and dilate to
a maximal diameter of approximately 2-3 times the size of the
diameter of the internal jugular vein (Fig. 3A,B) (Gittenberger-De
Groot et al., 2004). In Tie1neo/neo and Tie1–/– embryos at E13.5, the
maximal diameter of the jugular lymphatic sacs was approximately
10 times the size of the diameter of the internal jugular vein (Fig.
3D,F,G). This phenotype persists until at least E15.5 (data not
shown).
Fig. 3. Tie1 attenuation leads to dilation of jugular lymph sacs at
E13.5. (A-F) Transverse sections through the jugular region of wild-type
littermates (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 increase
in size of lymph sacs relative to the jugular vein and carotid artery in the
hypomorphic (D) and mutant (F) embryos and a thickened dermis and
subcutaneous layer (double-headed arrows) in the hypomorphic (C) and
mutant (E) embryonic skin when compared with the wild-type
littermates (A). (G) Quantification of lymph sac area, normalized to
jugular vein area in hypomorphic embryos, mutant embryos and their
wild-type littermates at E13.5. n>6 embryos per genotype. *, P<0.05.
jls, jugular lymph sacs; jv, jugular vein. Scale bars: 200 mm.
5H,J) and Tie1–/– (Fig. 5I,J) embryos were increased by 44.2% and
53.5%, respectively. These results suggest that an increased
production of LECs at the initiation of lymphangiogenesis
contributes to the dilated lymph sacs seen in the Tie1 hypomorphic
and mutant mice.
To evaluate whether Tie1 attenuation resulted in a global defect
in endothelial differentiation, we used dual immunoflourescence at
E11.5 (Fig. 5D-F) and E12.5 (see Fig. S2 in the supplementary
material) with specific vascular EC markers – Pecam1, CD34,
Icam1, endoglin or VE-cadherin and Prox1. We observed no
difference among wild-type, Tie1neo/neo and Tie1–/– embryos in the
expression of these five markers in vascular endothelium or at the
dorsal part of the cardinal vein, the site where lymphatic EC
differentiation occurs during embryogenesis. Thus, the abnormal
dilated lymph sac defects in Tie1neo/neo and Tie1–/– embryos could
not be ascribed to a primary endothelial defect.
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
indicate representative proliferative Prox1-positive lymphatic endothelial
nuclei in the wall of the cardinal vein and jugular lymph sac areas. cv,
cardinal vein; ls, lymph sac. Scale bar: 100 mm. (E) The percentage of
proliferative Prox1-positive cells in Tie1neo/neo embryos (gray bars) was
significantly higher than that in wild-type littermates at E11.5 and at
E12.5 (*, P<0.05), but no difference was seen at E13.5. Results
represent the means ± the s.d. of 5 embryos per group.
Tie1 attenuation leads to an increase in lymphatic
endothelial cell apoptosis
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
detected no significant difference in the percentage of apoptotic
LECs in the wall of the cardinal vein and jugular lymph sac areas
between wild-type (E11.5, 3.6±0.5%; E12.5, 3.2±0.8%) and
Tie1neo/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, we
identified an increase in apoptosis in LECs of hypomorphic
embryos. Only 2.5±0.6% TUNEL-positive cells were detected in
the lymphatic endothelium of the wild-type jugular lymphatic sacs
(Fig. 6A-C,G), whereas we observed 5.7±0.9% TUNEL-positive
LECs in the corresponding area of hypomorphic embryos (Fig.
6D-F,G). Interestingly, almost no EC apoptosis was seen in the
jugular vein or arteries of wild-type or hypomorphic embryos.
Taken together, these results demonstrate that reduction of Tie1
during embryonic development leads to a specific increase in
lymphatic, but not venous, EC apoptosis at later stages of
lymphangeogenesis.
DEVELOPMENT
Tie1 and lymphatic development
1290 RESEARCH ARTICLE
Development 137 (8)
Fig. 5. Tie1 attenuation leads to an increased lymphatic
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).
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–/– embryos
exhibit dilated jugular lymph sacs at E11.5 and at E12.5. cv, cardinal
vein; da, dorsal aorta; ls, lymph sac. Scale bars: 100 mm. Note: the
lymphatic marker Vegfr3 has weak expression in blood vessels at E12.5.
(J) Quantitative analysis of the number of Prox1-positive nuclei in the
cardinal vein and jugular lymph sac areas of embryos at E10.5 to E12.5.
*, P<0.05.
Abnormal dermal lymphatic pattern in Tie1
mutant embryos
We next performed wholemount immunohistochemistry to examine
the development of dermal lymphatic vasculature in Tie1neo/neo
embryos at E13.5. In wild-type embryos, a few, but clear, Vegfr3positive lymphatic vessels (Fig. 7A) and an extensive network of
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
the null mutant embryos (Fig. 7C,F) were more severe compared
with those in the hypomorphic embryos. Importantly, wholemount
staining of E13.5 forelimb skin for Pecam1 revealed a relatively
normal blood vessel pattern in the hypomorphic embryos and only
mildly dilated blood vessels in the null embryos (Fig. 7G-I),
indicating a preferential effect on lymphatic vessels. These results
suggested that the severity of lymphatic defects in Tie1 mutant
embryos is dependent on the dosage of Tie1 and that the
development of lymphatic vasculature is more sensitive to Tie1
reduction than the development of blood vasculature.
To further investigate network formation of blood vessels and
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
E14.5, the Vegfr3-positive lymphatic capillary network was detected
in wild-type control embryos, whereas lymphatic vessels were
disorganized and mildly dilated in Tie1neo/neo embryos; at E15.5,
lymphatic vessels in Tie1neo/neo embryos were dilated and lymphatic
vessels started to regress in some areas, and compared with the
phenotype in Tie1neo/neo embryos, the lymphatic defects in Tie1–/–
DEVELOPMENT
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.
Fig. 7. The severity of the dermal lymphatic defects in Tie1
mutant 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 lymphatic
network in the skin around the ear is normal in wild-type embryos (A),
but dilated and disorganized in Tie1neo/neo embryos (B), and even more
disrupted in Tie1–/– embryos (C). Similarly, a normal network of
lymphatic capillaries was detected in the forelimb skin of wild-type
embryos (D) and an irregular lymphatic network with dilated and
disorganized lymphatics was observed in the corresponding regions of
Tie1neo/neo (E) and Tie1–/– (F) embryos, respectively. (G-I) Forelimbs of
wild-type (G), Tie1neo/neo (H) and Tie1–/– (I) embryos at E13.5 were
labeled with Pecam1 antibody and the blood vessel pattern appeared
normal. Arrowheads indicate the diameter of the lymphatic vessels.
Scale bars: 250 mm.
embryos were even more severe. This was confirmed by Lyve1
immunoreactivity 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 Tie1
hypomorphic embryos was even more evident: Vegfr3-positive
lymphatic vessels were dramatically dilated in some areas or
severely disrupted in other areas (see Fig. S6A-C in the
supplementary material). At E17.5 (Fig. 9; see Fig. S6D-F in the
supplementary material), the Lyve1-positive lymphatic networks
were dense and well organized in wild-type embryos but were
markedly dilated, disorganized and irregular in Tie1 hypomorphic
embryo lymphatics. In addition, most of the wild-type lymphatic
capillaries were interconnected and only a few blind beginning
lymphatic capillaries were detected. By contrast, the formation of
lymphatic capillary networks was impaired in the head skin of the
hypomorphic mice. The skin in the hypomorphic embryos exhibited
incomplete network patterning with an increased number of blind
beginnings of lymphatics. Some regions were largely bereft of
lymphatics (Fig. 8B,F; see Fig. S5 and Fig. S6 in the supplementary
material), indicating regression of the developing lymphatic vessels.
To quantify the effect of Tie1 attenuation on lymphatic vessel
profiles, we measured lymphatic vessel area, density and diameter
(Fig. 8M-O). These studies documented an increase in vessel
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).
By contrast, blood vascular patterning was relatively unaffected
in 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 patterning
of the lymphatic vasculature without affecting the non-lymphatic
vasculature, suggesting that lymphatic dysfunction contributes to the
edema seen in the Tie1 hypomorphic and mutant mice. This finding
also supports the hypothesis that developing lymphatics are more
sensitive to Tie1 reduction than the arterial or venous vasculature.
Lymphatic function is impaired in Tie1neo/neo
embryos
To assess whether structural defects seen in dermal lymphatic
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
rapid in the collecting lymphatic vessels and network of capillaries
in wild-type mice at E17.5 (Fig. 9A). However, the transport
function of lymphatic vessels in the hypomorphic embryos was
impaired, consistent with the severity of the subcutaneous edema
defect. As seen in Fig. 9B, FITC-dextran labeled only a few
disorganized lymphatic capillaries in the hypomorphic embryo that
had severe edema and localized hemorrhages at E17.5, indicating
attenuation and/or poor function of the lymphatic capillary network.
We obtained similar results using wild-type and hypomorphic
embryos at E18.5 (Fig. 9C).
Lymphatic patterning is abnormal in internal
organs of Tie1 hypomorphic embryos
It is known that lymphatic capillaries progressively cover the heart
surface and the diaphragm from E15 onwards (Yuan et al., 2002).
Wholemount staining of these hearts with Lyve1 or Vegfr3 antibody
revealed regression of the developing lymphatic vessels at the
surface of the heart in the hypomorphic embryos compared with
control littermates (see Fig. S9A-F in the supplementary material),
which was first observed at E16.5. The epicardial lymphatic vessels
in the hypomorphic embryos formed a primitive continuous network
but were thinner than those in wild-type control embryos. In
addition, the lymphatic vessels of the mutant embryos had begun to
DEVELOPMENT
Tie1 and lymphatic development
1292 RESEARCH ARTICLE
Development 137 (8)
regress in some areas. At E17.5, the entire lymphatic network was
disrupted in the hypomorphic embryos and by E18.5, only a few
single disrupted lymphatic vessels were detected on the surface of
the heart. In the central portion of the diaphragm at E16.5, some of
the larger collecting lymphatics in the hypomorphic embryos were
dilated and disorganized compared with wild-type embryos (see Fig.
S9G-L in the supplementary material). By E17.5 and E18.5, there
was a striking reduction in the number of lymphatics in the whole
diaphragm of the hypomorphic embryos. Furthermore, all of the
lymphatics were much thinner than those in wild-type control
embryos and the network was disrupted, indicating regression of the
developing lymphatics over the entire diaphragmatic surface. Taken
together, the Tie1neo/neo mice demonstrated abnormally patterned
lymphatic vessels in all internal organs examined.
DISCUSSION
Genetic studies in mice have demonstrated that Tie1 is required for
development and maintenance of the vascular system. Deletion of
Tie1 leads to embryonic lethality due to edema, hemorrhage and
microvessel rupture (Puri et al., 1995; Sato et al., 1995).
Interestingly, the timing of previously reported embryonic demise
at E13.5 correlated with the earliest time when lymphatic vessels can
be morphologically distinguished from the blood vessels. The
previous report (Sato et al., 1995) and our study showed that the first
visible defect in the Tie1 mutants is dorsal subcutaneous edema from
E13.5 onward. In this report, we have documented that Tie1 is
expressed in LEC progenitors at E10.5 and E11.5 before lymphatic
vessels are formed. This expression of Tie1 in the lymphatic
endothelium is maintained throughout embryonic development and
persisted in the postnatal animal. The early expression in the
lymphatic endothelium suggests a unique role for Tie1 in the
development and function of the lymphatic vessels.
The intronic insertion of a neo cassette causes aberrant splicing of
Tie1 transcripts, reducing the amount of mRNA encoding functional
Tie1 protein to ~20% of normal levels. This Tie1 hypomorphic allele
enabled us to demonstrate that a dosage-dependent reduction of Tie1
results in progressive defects in lymphatic patterning not seen in other
EC populations. Tie1 attenuation resulted in significantly enlarged
jugular lymphatic vessels. Consistent with structural defects seen in
the lymphatics, lymphatic function was also impaired in hypomorphic
embryonic skin. We detected no apparent lymphatic defects in the
heterozygous Tie1+/neo embryos at any developmental stage.
Interestingly, although the Tie1 hypomorphic embryos display
severe generalized edema, there was almost no apparent hemorrhage
during the entire period of embryonic development. A few embryos
developed hemorrhage at the tips of the tail or digits and this
occurred at a much later time point than the diffuse hemorrhage
observed in the complete null embryos. In addition, although
lymphatic defects were obvious in most of the hypomorphic
embryos, the pattern of blood vessels appeared relatively normal. In
fact, when we examined expression of the specific blood vascular
EC markers in the major blood vessels and in particular at the dorsal
part of the cardinal vein, the site where lymphatic EC differentiation
occurs during the initiation of lymphangiogenesis, we could detect
no differences in the pattern of expression between wild-type and
hypomorphic mutant embryos. These results further suggest that the
development of lymphatic vasculature is more sensitive to Tie1
reduction than the development of blood vasculature.
DEVELOPMENT
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:
⫻10 (A-F); ⫻40 (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.
Fig. 9. Tie1 attenuation leads to impaired lymphatic drainage
function. (A,B) Representative image of the subcutaneous collecting
lymphatic vessels (arrows) and lymphatic capillaries (arrowheads) using
fluorescence microscopy after intradermal FITC-dextran injection into
embryonic forelimbs at E17.5. Sites of injection are indicated by dashed
circles. The lymphatic network in Tie1neo/neo embryos (B) is disorganized
and uptake of FITC-dextran is attenuated when compared with that
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)
in wild-type versus Tie1neo/neo embryos at E17.5 and at E18.5.
*, P<0.05.
It is commonly thought that most of the lymphovascular system
in mice is of venous origin (Oliver, 2004; Oliver and Alitalo, 2005).
However, recent data argues for a dual origin of LECs in the mouse
with incorporation of mesenchymal lymphangioblast-derived cells
into the lining of lymph vessels during development providing a
minor contribution to the lymphatics (Buttler et al., 2006; Buttler et
al., 2008). Although our work does not directly address whether all
lymphatics are of venous origin or whether there is an additional
contribution from a mesenchymal cell population, the fact that we
observed similar abnormalities in both the central lymphatics as well
as in the dermis of the embryo would suggest that Tie1 is required
for the normal development of both the central lymphatics and the
peripheral lymphatics.
Jugular lymphatic sac dilation was a prominent feature in the Tie1
mutant embryos. Attenuation of Tie1 leads to an increased LEC
production before mid-gestation in the Tie1 hypomorphic and
mutant mice. Consistently, we detected significantly more
proliferative LECs in the cardinal vein and jugular lymph sac areas
of Tie1 hypomorphic and mutant embryos at E11.5 and E12.5, when
specification of lymphatic vasculature occurs (Francois et al., 2008).
Although the proliferation rate of LECs in jugular lymphatic sacs of
Tie1 hypomorphic and mutant embryos at E13.5 returns to normal,
the absolute total number of proliferative LECs is still higher than
that in wild-type embryos because the absolute number of LECs is
higher (Fig. 5J).
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
a collecting vessel phenotype. Furthermore, dermal lymphatic
vessels in Ang2–/– pups prematurely recruit smooth muscle cells
and 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 ligandindependent signal transduction pathways have been characterized
(McCarthy et al., 1999; Yabkowitz et al., 1999). However, Tie1
phosphorylation can be induced by overexpression of multiple
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
(Marron et al., 2000) but the cellular function of heterodimerization
remains to be determined. As Tie1 is expressed in the endothelium
of the same lymphatics as Ang2 during embryogenesis and
enhanced Tie1 mRNA expression occurs together with Ang2 mRNA
in the adults (Gale et al., 2002; Iljin et al., 2002), it is tempting to
speculate that Tie1 might be modulating Ang2 function via
interaction with the Tie2 receptor in the lymphatic ECs as has been
documented in cultured ECs (Yuan et al., 2007) and endothelial
progenitor cells (Kim et al., 2006). Interestingly, investigators have
recently shown that Vegf can cause a 4-fold increase in
phosphorylation of Tie2 that is independent of angiopoietin
expression but dependent on proteolytic cleavage of Tie1 and
subsequent transphosphorylation of Tie2, further supporting the
possibility of Tie1 modulation of Tie2 signaling (Singh et al., 2009).
However, the expression of Tie2 and its role in LEC development
has not been well defined. Adult Lyve1-positive lymphatic
vessels have previously been shown to express Tie2 by
immunofluorescence labeling of mouse ear skin and the small
intestine using an antibody against Tie2 (Morisada et al., 2005;
Tammela et al., 2005). However, other groups failed to detect GFP
expression by lymphatic vessels in the adult (Dellinger et al., 2008)
or embryos (Srinivasan et al., 2007) from Tie2-GFP transgenic mice
using either immunohistochemical (GFP) analyses or in situ
hybridization. One possible explanation for this discrepancy is that
the regulatory elements controlling the expression of Tie2 by
DEVELOPMENT
Tie1 and lymphatic development
lymphatic vessels are not included in the Tie2-GFP transgenic
construct. Further delineation of the expression pattern of Tie2
should resolve this discrepancy.
In summary, it is now evident that the Tie receptors and their
ligands demonstrate context-dependent regulation of vascular
remodeling (Eklund et al., 2006) and our work suggests a unique
dosage-dependent role for Tie1 in lymphatic ontogeny. Whether
Tie1 signals independently or in association with Tie2 is a focus of
ongoing investigation. However, the development of a
hypomorphic, conditional Tie1 allele in combination with the
available lymphatic-specific Cre deletor lines (Srinivasan et al.,
2007) should provide the necessary reagents for the temporal- and
lymphatic-specific deletion required for further investigation.
Acknowledgements
We thank Dr Lawrence Price (Vanderbilt University) for providing the antibodies
against Prox1 and valuable discussions and Dr Christopher Brown (Vanderbilt
University) for critical reading of the manuscript. This work was supported by
NIH grants R01 HL086964 (S.B.) and DK038517 and a grant-in-aid from the
March of Dimes. Deposited in PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.043380/-/DC1
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Tie1 and lymphatic development