957
Development 119, 957-968 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
Expression of tie-2, a member of a novel family of receptor tyrosine
kinases, in the endothelial cell lineage
Harald Schnürch and Werner Risau
Max-Planck-Institut für Psychiatrie, Abteilung Neurochemie, Am Klopferspitz 18a, D-8033 Martinsried, Germany and MaxPlanck-Institut für physiologische und klinische Forschung, Abteilung Molekulare Zellbiologie, D-6350 Bad Nauheim,
Germany
SUMMARY
We are interested in the molecular mechanisms that are
involved in the development of the vascular system. In
order to respond to morphogenetic and mitogenic
signals, endothelial cells must express appropriate
receptors. To characterize endothelial cell-specific
receptors, we have concentrated on receptor tyrosine
kinases, because several lines of evidence suggested the
importance of controlled phosphotyrosine levels in endothelial cells. A strategy based on PCR amplification using
degenerate oligonucleotides and mouse brain capillaries
as mRNA source, led to the identification of a novel
receptor tyrosine kinase, which we designated tie-2. In
situ hybridization using a tie-2-specific probe revealed
an interesting spatial and temporal expression pattern.
The gene was expressed specifically in the endothelial
lineage. tie-2 transcripts were present in endothelial cell
precursors (angioblasts) and also in endothelial cells of
sprouting blood vessels throughout development and in
all organs and tissues so far examined. tie-2 was downregulated in the adult. Because of the unusual combination of immunoglobulin, EGF-like and fibronectin type
III domains in the extracellular portion of tie-2 which is
shared by TEK and tie, these molecules may be considered members of a new family of receptor tyrosine
kinases. Signal transduction via this new class of tyrosine
kinases could lead to a better understanding of the
molecular mechanisms of blood vessel formation.
INTRODUCTION
of endothelial cells in vitro and gives rise to hemangiomas
in chimeric mice in vivo (Williams et al., 1988; Montesano
et al., 1990). Polyoma middle T oncogene binds to and
thereby constitutively activates cytoplasmic tyrosine kinases
of the src family. Thus, in endothelial cells, tyrosine phosphorylation seems to play a critical role in the regulation of
proliferation, invasion and morphogenesis.
Since we are interested in the molecular mechanisms of
growth control and differentiation in endothelial cells, these
results prompted us to identify the genes encoding tyrosine
kinases in endothelial cells. We amplified DNA by the polymerase chain reaction using degenerate primers deduced
from conserved regions of receptor tyrosine kinases. Most
importantly, capillary fragments were isolated from
postnatal day 4-8 murine brains as a source of mRNA
because at this time sprouting and proliferation of endothelial cells is highest in this organ (Robertson et al., 1985).
The other rationale behind this strategy was to avoid the
cloning of tyrosine kinases such as FGF receptors that are
abundantly expressed by capillary endothelial cells in tissue
culture but not in vivo (Heuer et al., 1990; Peters et al.,
1992). Recently we have reported the successful application
of this strategy by the cloning and characterization of the
flk-1 receptor tyrosine kinase (Millauer et al., 1993). Here
Signal transduction by receptor and cytoplasmic tyrosine
kinases is a process of critical importance in cellular proliferation and differentiation. Phosphorylation on tyrosine of
cellular proteins has been detected in vivo using anti-phosphotyrosine antibodies. Proteins phosphorylated on tyrosine
were abundant during embryonic development but
decreased in adult tissues. Epithelial and endothelial cells
were found to be the cell types that were most prominently
labeled by the anti-phosphotyrosine antibodies (Maher and
Pasquale, 1988; Takata and Singer, 1988; Maher, 1991).
Proliferation and invasion of endothelial cells are crucial
events during the development of the vascular system.
Factors probably involved in the processes of vasculogenesis and angiogenesis that lead to the formation of a vascular
network (Risau, 1991) have been shown to bind with high
affinity to and activate receptor tyrosine kinases. In an in
vitro system of angiogenesis, molecules that regulate
tyrosine phosphorylation were found to modulate the
invasion and tube formation of endothelial cells (Montesano
et al., 1988; Montesano and Orci, 1985). Furthermore, the
expression of the polyoma middle T oncogene in endothelial
cells leads to aberrant growth and morphogenetic behavior
Key words: angiogenesis, receptor tyrosine kinase, endothelial
cells, angioblasts, mouse development, vascular development
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H. Schnürch and W. Risau
we have analyzed a different receptor tyrosine kinase,
named tie-2, that was isolated by this screening.
MATERIALS AND METHODS
Animals and tissues
Balb/c mice were mated overnight and the morning of vaginal plug
detection was defined as 0.5 day of gestation. Organs were
removed, frozen immediately in liquid nitrogen and stored at
−80°C. Capillary fragments from pooled P4-P8 mice brains were
prepared according to Risau et al. (1990). For in situ hybridization
and immunohistochemistry, whole embryos or organs were fixed
for 12 hours in freshly prepared 4% paraformaldehyde in PBS at
4°C, rinsed for 24-48 hours in 0.5 M sucrose in PBS at 4°C,
embedded in TissueTek (Miles) and stored frozen at −80°C.
RNA extraction and analysis
Total cytoplasmic RNA was isolated according to the method of
Chomczynski and Sacchi (1987). Enrichment for poly(A)+-containing fractions was achieved by oligo(dT) chromatography using
push columns (Stratagene). Aliquots of poly(A)+ RNA were electrophoresed in agarose gels containing 0.66 M formaldehyde and
transferred to Zeta-Probe membrane (Bio Rad) in 20× SSPE.
Hybridizations were performed overnight in 0.5 M sodium
phosphate buffer, 5% SDS, 1% BSA, pH 7.5 at 68°C with 1×106
cts/minute/ml of probe, which had been labelled with 32P-dCTP
according to the protocol of a random-primed DNA-labeling kit
(Boehringer Mannheim). Membranes were washed under highstringency conditions at 68°C in 0.1×SSPE, 0.5% SDS and autoradiographed at −80°C on Fuji films. Poly(A)+ RNA from brain
capillary fragments was isolated using a QuickPrep Micro mRNA
purification kit from Pharmacia.
PCR and cDNA cloning
Capillary poly(A)+ RNA was reverse transcribed and aliquots of
the cDNA were used as templates in PCR reactions. In addition to
those described by Wilks (1989), the following oligonucleotides
were used as primers: 5′-C A C/T C G I G A C/T C/T T I G C I
G C I A/C G-3′, 5′-A C/T I C C I A A/C I C/G A/T C C A I A C
A/G T C-3′ (I stands for inosine). The amplification products were
separated in acrylamide gels. Fragments of the expected size were
purified, labeled and used as probes to screen a random hexanucleotide-primed cDNA library constructed with a Time Saver
cDNA Synthesis Kit (Pharmacia). cDNA fragments were
subcloned into Bluescript vectors (Stratagene). Sequencing was
done by using nested oligonucleotide primers in combination with
a 373A DNA Sequencer (Applied Biosystems) and with the conventional Sequenase System (USB).
In situ hybridization
Preparation of tissue sections and in situ hybridization with singlestranded DNA probes or single-stranded RNA probes was
performed as described by Schnürch and Risau (1991). The probe
for all hybridization experiments was derived from a 1179 bp DNA
fragment encoding a portion of the putative tie-2 protein from
amino acid 419 to amino acid 812.
Immunohistochemistry
Embedded embryos and whole organs were sectioned on a Leitz
cryostat. 8 µm sections were mounted on organosilane-treated
slides, dried overnight under vacuum and stored desiccated at
−80°C. Sections were brought to room temperature, rehydrated in
PBS for 5 minutes and incubated in 0.1% H2O2 in methanol for 15
minutes. After washing three times in PBS for 5 minutes each,
unspecific antibody binding was blocked by application of 20%
normal goat serum in PBS for 20 minutes. Sections were washed,
incubated with a rat monoclonal antibody against mouse PECAM
(a generous gift from Dr E. Dejana) for 1 hour, washed again and
then incubated with a biotinylated goat anti-rat IgG (Dianova) for
1 hour. Color development was performed with a Vectastain ABC
kit (Vector Laboratories) according to the vendor´s protocol.
Sections were slightly counterstained with toluidine blue, dehydrated and mounted.
RESULTS
cDNA cloning and structure of the tie-2 protein
Our approach for the isolation of receptor tyrosine kinases
expressed in endothelial cells of sprouting blood vessels
involved the following steps. First, we purified mRNA from
capillaries of pooled P4-P8 mice brains. A portion of the
mRNA was reverse transcribed and used in PCR reactions
with degenerate primers deduced from conserved protein
regions of tyrosine kinases. Gel-purified reaction products
of the expected size were radiolabelled and used directly as
hybridization probes to screen a cDNA library constructed
from the remainder of the capillary mRNA.
Partially sequencing the inserts from positive phages
revealed that 13 of the cDNAs were derived from one
mRNA species. The longest of these 13 cDNAs, a fragment
of 4640 bp, was sequenced completely. It contained a long
open reading frame encoding a protein of 1123 amino acid
residues. This deduced polypeptide has all features of a
receptor tyrosine kinase: an amino-terminal signal sequence
followed by a long extracellular domain, a single hydrophobic transmembrane region and a cytoplasmic portion that
contains a tyrosine kinase domain (Fig. 1).
A survey for homologous proteins revealed that the
predicted protein is most closely related to the two recently
identified tyrosine kinases tie and tek (Partanen et al., 1992;
Dumont et al., 1992). With the exception of two residues,
the protein sequence published for tek is identical to the
intracellular part of our polypeptide, from position 823 to
the carboxy-terminal residue 1123. Both an extracellular
domain and a transmembrane region are missing in the tek
polypeptide (Fig. 2). It is therefore possible that tek represents a partial sequence or that the mRNA encoding tek is
the result of an alternative splice event, which results in the
production of a cytoplasmic tyrosine kinase. Comparison of
our protein with tie reveals a high degree of similarity especially in the cytoplasmic part (76% sequence identity). In
the extracellular domain and the transmembrane region, the
similarity is less pronounced (33% and 37% respectively;
Fig. 2). We will therefore refer to our protein as tie-2. While
this manuscript was in preparation, Ziegler et al. (1993)
reported the cloning of a cDNA whose translation product
is a receptor tyrosine kinase with an overall similarity of
more than 90% when compared to tie-2 (Fig. 2). It is
therefore likely that this protein, which they called TEK, is
the human homolog of tie-2. The intracellular part of tie-2
is also related to the product of the human ret protooncogene and to FGF receptors (Takahashi et al., 1989; Partanen
et al., 1991; Stark et al., 1991). The similarity to both is
about 43%. The intracellular part can be divided into three
characteristic regions: the juxtamembrane sequence, the
catalytic domain and the cytoplasmic tail. The kinase
tie-2 expression during vascular development
A
959
B
Fig. 1. Deduced amino acid sequence and structure of tie-2. (A) Amino acid sequence of tie-2 in single letter code. The potential signal
sequence cleavage site is indicated by an arrowhead. Black dots mark the two cysteine residues, possibly involved in sulfhydryl bonding
of the immunoglobulin domain. The three EGF-like repeats are boxed. The three fibronectin type III domains are underlined. The
transmembrane region is given in bold face letters. The tyrosine kinase domain is indicated by shaded boxes. The RGD triplet is marked
by an asterisk. (B) Schematic diagram of the structure of tie-2. Ig, immunoglobulin domain; EGF, EGF-like repeats; FN, fibronectin type
III domains; Kinase, tyrosine kinase domain; KI, kinase insertion.
domain, which is split by an 14 amino acid insertion
contains the GxGxxG consensus sequence that is part of the
ATP binding site. Typical tyrosine kinase motifs like
HRDLAARN and DFGL are present.
Within the extracellular part, the FASTA program
detected homologies to proteins including TAN-1 (Ellisen
et al., 1991), Xotch (Coffman et al., 1990), Laminin (Sasaki
et al., 1988), Delta (Vässin et al., 1987) and Perlecan
(Noonan et al., 1991) (around 30% sequence identity). The
structural basis for these homologies are three EGF-like
repeats in the center of the extracellular portion of tie-2 (Fig.
1). EGF-like repeats consist of 30 to 40 amino acid residues
often found in the extracellular parts of membrane-bound
proteins or secreted proteins (Davies, 1990). A common
feature of these domains are six conserved cysteine residues,
which are known to be involved in disulfide bonds. The
three EGF domains in tie-2 contain two additional cysteine
residues at the carboxy-terminal end of each repeat.
The central EGF-like repeats are flanked by two different
structural motifs, a single amino-terminal immunoglobulin
(Ig) domain and a carboxy-terminal triplet of fibronectin
(FN) type III domains. Although the Ig domain does not
exhibit clear sequence similarities to other known proteins
(except for tie and TEK), the Ig domain has the conserved
features of a C2-set domain including typical residues surrounding the first cysteine residue and the canonical GxYxC
found at the second cysteine residue (Williams and Barclay,
1988).
The three fibronectin type III (FN III) domains are most
closely related to FN III repeats present in the proteintyrosine phosphatases Delta (Krueger et al., 1990), LAR
(Streuli et al., 1988) and DLAR (Streuli et al., 1989) and to
FN III repeats of the axonal glycoprotein TAG-1 (Furley et
al., 1990) (around 20% sequence identity). These domains
have been described as units of approximately 90 amino
acids containing hydrophobic residues at characteristic
positions. In addition, a total of nine potential N-glycosylation sites are located in the extracellular part of tie-2.
tie-2 expression in brain capillaries
Our aim was to isolate receptor tyrosine kinases that are
expressed in blood vessels during brain angiogenesis. In
order to check the validity of the approach, we compared the
amount of tie-2 in mRNA from P4-P8 brain capillaries with
total P4 brain by northern analysis. To avoid artifactual
results, we used the part of the cDNA that encodes the less
conserved regions of the protein, namely the three FN III
domains, the transmembrane region and the juxtamembrane
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H. Schnürch and W. Risau
Fig. 2. Amino acid sequence comparison of tek, TEK and tie with tie-2. Amino acid residues identical to tie-2 are represented by (−).
Gaps are indicated by (.).
tie-2 expression during vascular development
Fig. 3. Northern analysis of tie-2 expression in brain capillaries
and total brain tissue from postnatal day 4 (P4) mice. A single
transcript of approximately 4.7 kb, highly enriched in the capillary
fraction was detected.
portion. Fig. 3 demonstrates the presence of a 4.7 kb mRNA
highly enriched in the capillary fraction. We could also
detect this single mRNA in organs like brain, kidney and
heart (data not shown).
tie-2 expression during brain development
Investigation of the spatial and temporal expression profile
of tie-2 during brain development was performed by in situ
hybridization. When we analyzed adult brain, P4 brain,
E12.5 brain and E8.5 neuroectoderm, it became evident that
tie-2 mRNA is exclusively synthesized in the vasculature of
these tissues (Fig. 4A-H). No other brain components were
labelled. In P4 brain, tie-2 expression was detected in capillaries that are about to invade the neural tissue as well as
in vessels that have already reached deeper layers of the neuroectoderm. High magnifications in Fig. 4G,H clearly show
the high concentration of silver grains over the vascular
perikarya. In addition to capillaries, meningeal blood vessels
and the choroid plexi were also found to synthesize tie-2
mRNA at comparable levels (Figs 4, 6B). At E12.5 the
overall expression pattern was virtually identical to that
observed at P4, although the density of labelled structures
in E12.5 brain was reduced (Fig. 4C,D). This observation
correlates with the less extensive vascularization of the
embryonic brain (Bär, 1980). No hybridization signals were
observed in the neuroectoderm of E8.5 embryos, because at
that stage this tissue is still avascular (Fig. 7). In the adult
brain, tie-2 expression was detectable, although it seemed to
be reduced when compared to postnatal or embryonic
stages. Hybridization signals persisted over larger vessels
especially those of the meninges (Fig. 4A,B). The synthesis
of tie-2 mRNA in smaller vessels and capillaries was barely
detectable.
Endothelial cell-specific expression of tie-2
To identify the cellular source of tie-2 mRNA, we performed
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in situ hybridization and immunohistochemistry on adjacent
sections. The pattern of tie-2 hybridization signals was
compared with the immunohistochemical staining of a monoclonal antibody against the endothelial cell-specific
adhesion molecule PECAM(CD31) (Newman et al., 1990).
Fig. 5 shows a representative example of one such experiment. The antibody stains the continuous layer of endothelial cells surrounding the lumen of a medium-sized blood
vessel in the head region of an E12.5 embryo. On the
adjacent section, the tie-2-specific probe labels the vessel in
an identical way. These results, together with the northern
hybridization signal detected in RNA from a capillary
fraction highly enriched for endothelial cells, provide strong
evidence for endothelium-specific expression of tie-2. Fig.
6 gives a survey on the coexpression of tie-2 and PECAM
in several organs and tissues. It is clear that tie-2 mRNA is
present in endothelial cells all over the body. Strong hybridization signals were associated with the heart endocardium
as well with the myocardial blood vessels (Fig. 6E,F). The
same holds true for the endothelium of the dorsal aorta (Fig.
6C,D), the intersomitic vasculature, the vessels surrounding
the lung bronchia and the capillaries perforating the spinal
cord (Fig. 6H,D). In summary, tie-2 gene expression seems
to be a general feature of endothelial cells.
tie-2 expression during early stages of
development
The possible role of tie-2 in early stages of vascular development was investigated by in situ hybridization studies
with E8.5 sections. The probe detected tie-2 mRNA in the
mesodermal component of the yolk sac (Fig. 7). This finding
is of particular interest, because it is in the mesodermal
component of the yolk sac that the first signs of blood vessel
development are evident. Clusters of mesenchymal cells
form the so-called blood islands. At the margin of these
aggregates cells, the so-called angioblasts, adopt an endothelial-like phenotype, whereas in the center cells differentiate into embryonic hemoblasts. Fig. 7 demonstrates that the
peripheral angioblasts synthesize high levels of tie-2
mRNA.
Within the embryo proper, expression was seen in the
anlagen of the vascular system, e. g. in the developing endocardium, the dorsal aortae and the cardinal veins (Fig.
7A,B). It is likely that these signals stem from intraembryonic endothelial cell precursors, which are present in these
structures at that stage. tie-2 expression could also be
detected in the allantois and in blood vessels of the maternal
decidua (data not shown).
DISCUSSION
We report here the isolation and characterization of tie-2, an
endothelium-specific receptor tyrosine kinase with an
unusual topology. So far only three other tyrosine kinases
with similar features have been described, namely tie, tek
and TEK. Although no studies on the developmental
expression are available, tie mRNA has been detected in
endothelial cells (Partanen et al., 1992). The spatial and
temporal expression pattern of tek seems to overlap largely
with that of tie-2. No in situ hybridization data were
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tie-2 expression during vascular development
963
Fig. 5. Colocalization of tie-2 expression with immunostaining for PECAM(CD31). Adjacent section of E12.5 embryos were hybridized
with a tie-2 probe (A) or stained with an antibody recognizing PECAM (CD31) (B). Arrowheads indicate expression of tie-2 in the
endothelial cell layer of a medium-sized blood vessel from the head region. Bar represents 10 µm.
presented for TEK (Ziegler et al., 1993). However, because
TEK seems to be the human homolog of tie-2, the spatial
and temporal expression profiles of both genes may be
similar if not identical. Obviously, there is a family of structurally related molecules synthesized by endothelial cells.
The question arises how many members this family may
have? Dumont et al. (1992) detected several mRNA species
by northern analysis in embryonic heart tissue hybridizing
with a tek probe. These findings can be explained by differential, perhaps organ-specific splice events that could
lead to the production of truncated and/or cytoplasmic
tyrosine kinases. Alternatively, by using a cDNA encoding
the highly conserved catalytic domain as a probe, they may
have detected closely related molecules. This possibility
cannot be excluded, because we used the less conserved part
of the tie-2 cDNA and detected only one single mRNA
species. It will therefore be interesting to see whether this
still small family will grow by additional members.
The fact that we could detect tie-2 mRNA in a capillary
fraction highly enriched for endothelial cells gave us the first
hint for an endothelium-specific expression pattern of this
gene. This finding confirmed the suitability of our experimental design. Instead of cultured endothelial cells, we used
freshly prepared capillary material from P4 mouse brain as
a mRNA source for the initial cloning step. There were two
reasons for this approach. First, the proliferation of capillaries is maximal during the first postnatal days in the rodent
Fig. 4. tie-2 expression during brain development. Sagittal
sections of adult brain (A,B), postnatal day 4 brain (C,D,G,H) and
embryonic day 12.5 brain (E,F) were hybridized with a tie-2
probe. Bright-field (A,C,E) and corresponding dark-field
micrographs (B,D,F) Arrowheads indicate expression in
capillaries and arrows indicate hybridization signals over
meningeal blood vessels. Higher magnification of a capillary
sprout at the dorsal surface of a postnatal day 4 telencephalon (G).
Higher magnification of a capillary in a deeper layer of the brain
(H). No other than the vascular elements of the brain were found
to synthesize tie-2 mRNA. LM, leptomeninges; LV, lateral
ventricle; TE, telencephalon; (A, B, C, D, E, F) bar represents 110
µm; (G, H) bar represents 10 µm;
brain (Robertson et al., 1985). Second, receptors for FGF,
although produced by endothelial cells in vitro, have not be
detected in capillary endothelial cells in vivo (Heuer et al.,
1990; Peters et al., 1992). Our in situ hybridization data on
embryonic, postnatal and adult tissue sections confirmed
that tie-2 is specifically expressed in endothelial cells. The
spatial expression pattern of tie-2 was found to be identical
to the immunohistochemical staining for the endothelial cell
marker PECAM(CD 31) (Newman et al., 1990).
The developing brain does not contain endogenous endothelial cell precursors (Noden, 1990). As a consequence, this
organ is vascularized by angiogenic sprouting from the
meningeal vascular plexus (Bär, 1980). The whole process
consists of a complex pattern of physiological events
involving sprouting, migration and proliferation of endothelial cells and finally their differentiation into tube-like
structures surrounded by a basal lamina. Due to its
expression pattern, tie-2 could be involved in any of this
events. tie-2 synthesis in the vascular plexus could reflect
the competence of endothelial cells to respond to an angiogenic stimulus. Expression both at the tip of an invading
capillary and in more advanced vessels would argue for a
participation in migration and proliferation. In the adult
brain, tie-2 seemed to be down regulated and was detectable
mainly in larger vessels of the meninges. Because endothelial cell turnover is low in the adult, persistent expression
of tie-2 would favor a role in the establishment and the maintenance of an endothelium-specific phenotype in later stages
of development. Since the vascular extracellular matrix also
changes during embryonic development (Risau and
Lemmon, 1988), tie-2 with its peculiar organization of extracellular domains may be involved in the regulation of endothelial cell-substratum adhesion.
It is unlikely that the multiple aspects of endothelial cell
physiology are controlled by a single receptor. Recently, we
have identified flk-1 as an endothelium-specific receptor
tyrosine kinase in an identical approach as described here
for tie-2 (Millauer et al., 1993). The temporal and spatial
expression pattern of flk-1, which is a receptor for vascular
endothelial cell growth factor (VEGF), seems to be similar,
if not identical to that found for tie-2. However, both
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H. Schnürch and W. Risau
Fig. 6
tie-2 expression during vascular development
965
Fig. 7. tie-2 expression at embryonic day 8.5. Transverse section of an E 8.5 embryo and adjacent yolk sac. Hybridization signals were
detectable in the endocardium, dorsal aorta, cardinal vein and the mesodermal (inner layer) of the yolk sac (A,B). Arrowheads indicate
hybridization signal over the marginal cells of an advanced stage blood island (C,D). Note the absence of hybridization signals in the
neuroectoderm. CV, cardinal vein (head vein); BI, blood island; DA, dorsal aorta; EN, endocardial tissue; TE, telencephalon; YS, yolk
sac. Bar, represents 50 µm.
receptors differ in topology, especially in their extracellular
domains. It is conceivable that a set of receptors, whose
composition changes in a time- and tissue-dependent
manner, specifies endothelial cell behaviour during blood
vessel development and maintenance.
Fig. 6. tie-2 expression in organs and tissues of E 12.5 embryos
and colocalization with immunostaining for PECAM (CD31).
Adjacent sections of E12.5 embryos were stained with an
antibody recognizing PECAM(CD31) (A,C,E,G) or hybridized
with a tie-2 probe (B,D,F,H). The expression pattern of tie-2 was
found to be identical to the staining pattern fort PECAM(CD31).
TE, telencephalon; LV, lateral ventricle; CP, choroid plexus of the
lateral ventricle; DA, dorsal aorta; SC, spinal cord; EN,
endocardium; MY, myocardium; BR, bronchus; SO, somite; Bar
represents 110 µm.
From transplantation studies in the quail-chick system, it
is well documented that the ability of endothelial cells to
form a blood vessel system is not due to an intrinsic genetic
program, but depends on environmental cues from the tissue
to be vascularized (Noden, 1988; Stewart and Wiley, 1981;
Ekblom et al., 1982). Given the final morphological heterogeneity of the vasculature, it is clear that recognizing and
responding to different external signals must be a central
aspect in endothelial cell biology. The tie-2 receptor is ubiquitously expressed in the endothelium of every organ and
tissue that we have examined. Apart from brain, it was
detected in heart, lung, kidney, liver and spinal cord. It is
therefore likely that tie-2 is involved in a very fundamental
signalling process, typical for the whole population of heterogeneous endothelial cells.
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The finding that tie-2 is expressed in E8.5 embryos
confirms this notion and suggests a role even at the early
phases of vascular development. Blood islands are mesodermal derivatives of the yolk sac with characteristic morphology. The appearance of a central cell cluster distinct
from the peripheral cell layer, is the first overt sign for the
separation of the hematopoietic and endothelial cell lineage.
We could detect tie-2 mRNA in the peripheral endothelial
cell precursors indicating that the gene could be involved in
the onset of angioblast differentiation. tie-2 expression was
also observed in yolk sac mesoderm outside of blood
islands. The significance of this observation is not clear.
However, one may speculate that tie-2 mRNA synthesis
reflects the segregation of endothelial cells and hematopoietic cells on a molecular level before this process is morphologically evident.
The origin and the differentiation of endothelial cells
within the embryo proper is less well understood.
However, from the transplantation studies mentioned
above, it is clear that endogenous endothelial cell precursors are present in most mesodermal tissues of the embryo.
The development of some intraembryonic blood vessels,
e.g. the dorsal aortae, has been described as an assembly
of in situ differentiating endothelial cells (Meier, 1980;
Pardanaud et al., 1987; Coffin and Poole, 1988; Pardanaud
et al., 1989). This mode of blood vessel formation, which
is called vasculogenesis, is mechanistically distinct from
angiogenic sprouting (Pardanaud et al., 1987, 1989; Risau
et al., 1988). We found tie-2 mRNA expressed in cells
assembled like a ring in the anlagen of the dorsal aortae.
The same pattern was found in the primordia of the
cardinal veins. Furthermore, tie-2 mRNA was present in
the developing endocardium of the heart and in scattered
mesenchymal cells, which were not associated with a particular structure. This expression profile could be indicative of the presence of intraembryonic endothelial cell precursors before and at the time of differentiation. In that
case, tie-2 would be an early marker for intra- and extraembryonic angioblasts and molecular probes for tie-2 would
be a valuable tool for studying the early events of vascular
development.
Endothelial cells are able to interpret external cues for
their correct assembly into a highly diverse blood vessel
system. The tie-2 receptor may be considered as part of a
signaling system allowing endothelial cells to respond to
soluble or matrix-bound factors and to communicate with
other cells. The exact role of tie-2 on a molecular level has
not been determined yet. The intracellular portion of the
deduced protein contains a typical tyrosine kinase domain.
The identification of substrates for phosphorylation and the
question whether there exists a endothelial cell-specific
signaling transduction pathway will be an area of future
research. In the extracellular portion of tie-2 is a mosaic of
immunoglobulin, EGF-like and fibronectin type III
domains. EGF-like repeats are present in numerous, apparently unrelated proteins. The functional significance of this
sequence motif is not obvious. Several copies can be
present in the extracellular part of membrane-bound
proteins or in polypeptides that are secreted. The three
EGF-like repeats in tie-2 show about 30% sequence similarity with TAN-1 (Ellisen et al., 1991) and Xotch
(Coffman et al., 1990), vertebrate homologues of the
Drosophila notch gene product. Tan-1 and Xotch are
integral membrane proteins, each containing 36 EGF-like
repeats in the extracellular part. The molecules are thought
to be involved in some sort of extracellular interaction. The
A chain of laminin, a glycosylated basal lamina protein,
possesses 15 EGF-like repeats, three of them having a 30%
sequence identity with the EGF-like triplet of tie-2 (Sasaki
et al., 1988). Unlike TAN-1, Xotch and Laminin, the EGFlike units in tie-2 (also tie and TEK) have eight instead of
six cysteine residues. It is an open question whether these
additional residues are also involved in disulphide bonding
and whether this has functional importance for the interaction of tie-2 to its yet unidentified ligand. In any event, to
our knowledge tie and tie-2 are so far the only receptor
tyrosine kinases known to have EGF-like repeats in their
extracellular parts.
In contrast to EGF-like repeats, immunoglobulin-like
domains are structural motifs present in the extracellular
portions of wide variety of receptor tyrosine kinases (Ullrich
and Schlessinger, 1990; Williams and Barclay, 1988). tie-2
has a single, amino-terminal Ig domain with the characteristics of a C2-set sequence. In general, the function of Igcontaining proteins can be described as mediating heterophilic and homophilic binding events. In the case of the
alpha-PDGF receptor, the three amino-terminal Ig domains
determine the binding specificity for the PDGF AA ligand
(Heidaran et al., 1990).
Fibronectin type III (FN III) repeats are also present in
the extracellular parts of receptor tyrosine kinases
including the insulin receptor, IGF-1 receptor (Ullrich et
al., 1986), sevenless (Norton et al., 1990), Eph (Hirai et
al., 1987), cek5 (Pasquale, 1991), axl (O’Bryan et al.,
1991) and ark (Rescigno et al., 1991). Originally described
as internal homologous units in the extracellular matrix
(ECM) protein fibronectin (Petersen et al., 1983), FN III
repeats have been identified in, for example, adhesion
molecules, cytokine receptors and collagens. In the tenth
FN III repeat of fibronectin, the tripeptide RGD was found
to be crucial for the interaction of the ECM protein with
integrins on the cell surface (Ruoslahti and Pierschbacher,
1986). The three FN III repeats of tie-2 are mostly related
to those found in protein-tyrosine phosphatases and in the
axonal glycoprotein TAG-1. A possible RGD cell attachment site is present between the amino-terminal Ig domain
and the three EGF-like repeats of tie-2. The significance of
this observation is unclear, because the tripeptide sequence
is neither in a FN III repeat context nor is it conserved in
tie and TEK. Interestingly, the ECM protein laminin
appears to mediate its effects on endothelial cells in part
by a RGD-containing sequence in the A chain (Grant et al.,
1989).
Taken together, tie-2 has significant similarities to ECM
proteins and adhesion molecules. Whether these homologies
indicate a role for this receptor tyrosine kinase in cell-cell
or cell-matrix interactions, or whether the ligand for tie-2 is
a diffusible molecule remains to be determined. Due to the
unusual topology of tie-2, the ligand could be a novel
molecule whose identification could lead to a more complete
understanding of the molecular mechanisms of blood vessel
formation.
tie-2 expression during vascular development
We would like to thank Susanne Wizigmann-Voos for collaboration, Dorothea Rocholl for technical assistance, Elisabeta Dejana
for the generous gift of the anti-PECAM antibody and Carolin and
Georg Breier for artwork.
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Note added in proof
Dumont et al. have recently reported a full-length tek
sequence that seems to be identical to tie-2 (Oncogene
(1993), 8, 1293-1301).