Expression of Tie-2, a Member of a Novel Family of Receptor Tyrosine Kinases, in the Endothelial Cell Lineage

Development 119, 957-968 (1993) 957 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 Max- Planck-Institut für physiologische und klinische Forschung, Abteilung Molekulare Zellbiologie, D-6350 Bad Nauheim, Germany

SUMMARY

We are interested in the molecular mechanisms that are lineage. tie-2 transcripts were present in endothelial cell involved in the development of the vascular system. In precursors (angioblasts) and also in endothelial cells of order to respond to morphogenetic and mitogenic sprouting blood vessels throughout development and in signals, endothelial cells must express appropriate all organs and tissues so far examined. tie-2 was down- receptors. To characterize endothelial cell-specific regulated in the adult. Because of the unusual combina- receptors, we have concentrated on receptor tyrosine tion of immunoglobulin, EGF-like and fibronectin type kinases, because several lines of evidence suggested the III domains in the extracellular portion of tie-2 which is importance of controlled phosphotyrosine levels in endo- shared by TEK and tie, these molecules may be consid- thelial cells. A strategy based on PCR amplification using ered members of a new family of receptor tyrosine degenerate oligonucleotides and mouse brain capillaries kinases. Signal transduction via this new class of tyrosine as mRNA source, led to the identification of a novel kinases could lead to a better understanding of the receptor tyrosine kinase, which we designated tie-2. In molecular mechanisms of blood vessel formation. situ hybridization using a tie-2-specific probe revealed an interesting spatial and temporal expression pattern. Key words: angiogenesis, receptor tyrosine kinase, endothelial The gene was expressed specifically in the endothelial cells, angioblasts, mouse development, vascular development

INTRODUCTION of endothelial cells in vitro and gives rise to hemangiomas in chimeric mice in vivo (Williams et al., 1988; Montesano Signal transduction by receptor and cytoplasmic tyrosine et al., 1990). Polyoma middle T oncogene binds to and kinases is a process of critical importance in cellular prolif- thereby constitutively activates cytoplasmic tyrosine kinases eration and differentiation. Phosphorylation on tyrosine of of the src family. Thus, in endothelial cells, tyrosine phos- cellular proteins has been detected in vivo using anti-phosphorylation seems to play a critical role in the regulation of photyrosine antibodies. Proteins phosphorylated on tyrosine proliferation, invasion and morphogenesis. were abundant during embryonic development but Since we are interested in the molecular mechanisms of decreased in adult tissues. Epithelial and endothelial cells growth control and differentiation in endothelial cells, these were found to be the cell types that were most prominently results prompted us to identify the genes encoding tyrosine labeled by the anti-phosphotyrosine antibodies (Maher and kinases in endothelial cells. We amplified DNA by the poly- Pasquale, 1988; Takata and Singer, 1988; Maher, 1991). merase chain reaction using degenerate primers deduced Proliferation and invasion of endothelial cells are crucial from conserved regions of receptor tyrosine kinases. Most events during the development of the vascular system. importantly, capillary fragments were isolated from Factors probably involved in the processes of vasculogene- postnatal day 4-8 murine brains as a source of mRNA sis and angiogenesis that lead to the formation of a vascular because at this time sprouting and proliferation of endo- network (Risau, 1991) have been shown to bind with high thelial cells is highest in this organ (Robertson et al., 1985). affinity to and activate receptor tyrosine kinases. In an in The other rationale behind this strategy was to avoid the vitro system of angiogenesis, molecules that regulate cloning of tyrosine kinases such as FGF receptors that are tyrosine phosphorylation were found to modulate the abundantly expressed by capillary endothelial cells in tissue invasion and tube formation of endothelial cells (Montesano culture but not in vivo (Heuer et al., 1990; Peters et al., et al., 1988; Montesano and Orci, 1985). Furthermore, the 1992). Recently we have reported the successful application expression of the polyoma middle T oncogene in endothelial of this strategy by the cloning and characterization of the cells leads to aberrant growth and morphogenetic behavior flk-1 receptor tyrosine kinase (Millauer et al., 1993). Here 958 H. Schnürch and W. Risau we have analyzed a different receptor tyrosine kinase, incubated with a rat monoclonal antibody against mouse PECAM named tie-2, that was isolated by this screening. (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 MATERIALS AND METHODS kit (Vector Laboratories) according to the vendor´s protocol. Sections were slightly counterstained with toluidine blue, dehy- Animals and tissues drated and mounted. 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 RESULTS - 80°C. Capillary fragments from pooled P4-P8 mice brains were prepared according to Risau et al. (1990). For in situ hybridization cDNA cloning and structure of the tie-2 protein and immunohistochemistry, whole embryos or organs were fixed Our approach for the isolation of receptor tyrosine kinases for 12 hours in freshly prepared 4% paraformaldehyde in PBS at expressed in endothelial cells of sprouting blood vessels 4°C, rinsed for 24-48 hours in 0.5 M sucrose in PBS at 4°C, involved the following steps. First, we purified mRNA from embedded in TissueTek (Miles) and stored frozen at - 80°C. capillaries of pooled P4-P8 mice brains. A portion of the RNA extraction and analysis mRNA was reverse transcribed and used in PCR reactions Total cytoplasmic RNA was isolated according to the method of with degenerate primers deduced from conserved protein Chomczynski and Sacchi (1987). Enrichment for poly(A)+-con- regions of tyrosine kinases. Gel-purified reaction products taining fractions was achieved by oligo(dT) chromatography using of the expected size were radiolabelled and used directly as push columns (Stratagene). Aliquots of poly(A)+ RNA were elec- hybridization probes to screen a cDNA library constructed trophoresed in agarose gels containing 0.66 M formaldehyde and from the remainder of the capillary mRNA. transferred to Zeta-Probe membrane (Bio Rad) in 20´ SSPE. Partially sequencing the inserts from positive phages Hybridizations were performed overnight in 0.5 M sodium revealed that 13 of the cDNAs were derived from one 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 mRNA species. The longest of these 13 cDNAs, a fragment according to the protocol of a random-primed DNA-labeling kit of 4640 bp, was sequenced completely. It contained a long (Boehringer Mannheim). Membranes were washed under high- open reading frame encoding a protein of 1123 amino acid stringency conditions at 68°C in 0.1´ SSPE, 0.5% SDS and autora- residues. This deduced polypeptide has all features of a diographed at - 80°C on Fuji films. Poly(A)+ RNA from brain receptor tyrosine kinase: an amino-terminal signal sequence capillary fragments was isolated using a QuickPrep Micro mRNA followed by a long extracellular domain, a single hydropho- purification kit from Pharmacia. bic transmembrane region and a cytoplasmic portion that PCR and cDNA cloning contains a tyrosine kinase domain (Fig. 1). Capillary poly(A)+ RNA was reverse transcribed and aliquots of A survey for homologous proteins revealed that the the cDNA were used as templates in PCR reactions. In addition to predicted protein is most closely related to the two recently those described by Wilks (1989), the following oligonucleotides identified tyrosine kinases tie and tek (Partanen et al., 1992; were used as primers: 5¢-C A C/T C G I G A C/T C/T T I G C I Dumont et al., 1992). With the exception of two residues, 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 the protein sequence published for tek is identical to the A/G T C-3¢(I stands for inosine). The amplification products were intracellular part of our polypeptide, from position 823 to separated in acrylamide gels. Fragments of the expected size were the carboxy-terminal residue 1123. Both an extracellular purified, labeled and used as probes to screen a random hexanu- domain and a transmembrane region are missing in the tek cleotide-primed cDNA library constructed with a Time Saver polypeptide (Fig. 2). It is therefore possible that tek repre- cDNA Synthesis Kit (Pharmacia). cDNA fragments were subcloned into Bluescript vectors (Stratagene). Sequencing was sents a partial sequence or that the mRNA encoding tek is done by using nested oligonucleotide primers in combination with the result of an alternative splice event, which results in the a 373A DNA Sequencer (Applied Biosystems) and with the con- production of a cytoplasmic tyrosine kinase. Comparison of ventional Sequenase System (USB). our protein with tie reveals a high degree of similarity especially in the cytoplasmic part (76% sequence identity). In In situ hybridization the extracellular domain and the transmembrane region, the Preparation of tissue sections and in situ hybridization with single- similarity is less pronounced (33% and 37% respectively; stranded DNA probes or single-stranded RNA probes was Fig. 2). We will therefore refer to our protein as tie-2. While performed as described by Schnürch and Risau (1991). The probe for all hybridization experiments was derived from a 1179 bp DNA this manuscript was in preparation, Ziegler et al. (1993) fragment encoding a portion of the putative tie-2 protein from reported the cloning of a cDNA whose translation product amino acid 419 to amino acid 812. is a receptor tyrosine kinase with an overall similarity of more than 90% when compared to tie-2 (Fig. 2). It is Immunohistochemistry therefore likely that this protein, which they called TEK, is Embedded embryos and whole organs were sectioned on a Leitz the human homolog of tie-2. The intracellular part of tie-2 cryostat. 8 mm sections were mounted on organosilane-treated is also related to the product of the human ret protoonco- slides, dried overnight under vacuum and stored desiccated at - 80°C. Sections were brought to room temperature, rehydrated in gene and to FGF receptors (Takahashi et al., 1989; Partanen et al., 1991; Stark et al., 1991). The similarity to both is 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, about 43%. The intracellular part can be divided into three unspecific antibody binding was blocked by application of 20% characteristic regions: the juxtamembrane sequence, the normal goat serum in PBS for 20 minutes. Sections were washed, catalytic domain and the cytoplasmic tail. The kinase tie-2 expression during vascular development 959

A 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 features of a C2-set domain including typical residues sur- contains the GxGxxG consensus sequence that is part of the rounding the first cysteine residue and the canonical GxYxC ATP binding site. Typical tyrosine kinase motifs like found at the second cysteine residue (Williams and Barclay, HRDLAARN and DFGL are present. 1988). Within the extracellular part, the FASTA program The three fibronectin type III (FN III) domains are most detected homologies to proteins including TAN-1 (Ellisen closely related to FN III repeats present in the protein- et al., 1991), Xotch (Coffman et al., 1990), Laminin (Sasaki tyrosine phosphatases Delta (Krueger et al., 1990), LAR et al., 1988), Delta (Vässin et al., 1987) and Perlecan (Streuli et al., 1988) and DLAR (Streuli et al., 1989) and to (Noonan et al., 1991) (around 30% sequence identity). The FN III repeats of the axonal glycoprotein TAG-1 (Furley et structural basis for these homologies are three EGF-like al., 1990) (around 20% sequence identity). These domains repeats in the center of the extracellular portion of tie-2 (Fig. have been described as units of approximately 90 amino 1). EGF-like repeats consist of 30 to 40 amino acid residues acids containing hydrophobic residues at characteristic often found in the extracellular parts of membrane-bound positions. In addition, a total of nine potential N-glycosyla- proteins or secreted proteins (Davies, 1990). A common tion sites are located in the extracellular part of tie-2. feature of these domains are six conserved cysteine residues, which are known to be involved in disulfide bonds. The tie-2 expression in brain capillaries three EGF domains in tie-2 contain two additional cysteine Our aim was to isolate receptor tyrosine kinases that are residues at the carboxy-terminal end of each repeat. expressed in blood vessels during brain angiogenesis. In The central EGF-like repeats are flanked by two different order to check the validity of the approach, we compared the structural motifs, a single amino-terminal immunoglobulin amount of tie-2 in mRNA from P4-P8 brain capillaries with (Ig) domain and a carboxy-terminal triplet of fibronectin total P4 brain by northern analysis. To avoid artifactual (FN) type III domains. Although the Ig domain does not results, we used the part of the cDNA that encodes the less exhibit clear sequence similarities to other known proteins conserved regions of the protein, namely the three FN III (except for tie and TEK), the Ig domain has the conserved domains, the transmembrane region and the juxtamembrane 960 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 961

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 experi- ment. 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. Fig. 3. Northern analysis of tie-2 expression in brain capillaries 6C,D), the intersomitic vasculature, the vessels surrounding and total brain tissue from postnatal day 4 (P4) mice. A single the lung bronchia and the capillaries perforating the spinal transcript of approximately 4.7 kb, highly enriched in the capillary cord (Fig. 6H,D). In summary, tie-2 gene expression seems fraction was detected. to be a general feature of endothelial cells. portion. Fig. 3 demonstrates the presence of a 4.7 kb mRNA tie-2 expression during early stages of highly enriched in the capillary fraction. We could also development detect this single mRNA in organs like brain, kidney and The possible role of tie-2 in early stages of vascular devel- heart (data not shown). opment was investigated by in situ hybridization studies with E8.5 sections. The probe detected tie-2 mRNA in the tie-2 expression during brain development mesodermal component of the yolk sac (Fig. 7). This finding Investigation of the spatial and temporal expression profile is of particular interest, because it is in the mesodermal of tie-2 during brain development was performed by in situ component of the yolk sac that the first signs of blood vessel hybridization. When we analyzed adult brain, P4 brain, development are evident. Clusters of mesenchymal cells E12.5 brain and E8.5 neuroectoderm, it became evident that form the so-called blood islands. At the margin of these tie-2 mRNA is exclusively synthesized in the vasculature of aggregates cells, the so-called angioblasts, adopt an endo- these tissues (Fig. 4A-H). No other brain components were thelial-like phenotype, whereas in the center cells differen- labelled. In P4 brain, tie-2 expression was detected in cap- tiate into embryonic hemoblasts. Fig. 7 demonstrates that the illaries that are about to invade the neural tissue as well as peripheral angioblasts synthesize high levels of tie-2 in vessels that have already reached deeper layers of the neu- mRNA. roectoderm. High magnifications in Fig. 4G,H clearly show Within the embryo proper, expression was seen in the the high concentration of silver grains over the vascular anlagen of the vascular system, e. g. in the developing endo- perikarya. In addition to capillaries, meningeal blood vessels cardium, the dorsal aortae and the cardinal veins (Fig. and the choroid plexi were also found to synthesize tie-2 7A,B). It is likely that these signals stem from intraembry- mRNA at comparable levels (Figs 4, 6B). At E12.5 the onic endothelial cell precursors, which are present in these overall expression pattern was virtually identical to that structures at that stage. tie-2 expression could also be observed at P4, although the density of labelled structures detected in the allantois and in blood vessels of the maternal in E12.5 brain was reduced (Fig. 4C,D). This observation decidua (data not shown). 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 DISCUSSION that stage this tissue is still avascular (Fig. 7). In the adult brain, tie-2 expression was detectable, although it seemed to We report here the isolation and characterization of tie-2, an be reduced when compared to postnatal or embryonic endothelium-specific receptor tyrosine kinase with an stages. Hybridization signals persisted over larger vessels unusual topology. So far only three other tyrosine kinases especially those of the meninges (Fig. 4A,B). The synthesis with similar features have been described, namely tie, tek of tie-2 mRNA in smaller vessels and capillaries was barely and TEK. Although no studies on the developmental detectable. expression are available, tie mRNA has been detected in endothelial cells (Partanen et al., 1992). The spatial and Endothelial cell-specific expression of tie-2 temporal expression pattern of tek seems to overlap largely To identify the cellular source of tie-2 mRNA, we performed with that of tie-2. No in situ hybridization data were 962 H. Schnürch and W. Risau 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 mm. presented for TEK (Ziegler et al., 1993). However, because brain (Robertson et al., 1985). Second, receptors for FGF, TEK seems to be the human homolog of tie-2, the spatial although produced by endothelial cells in vitro, have not be and temporal expression profiles of both genes may be detected in capillary endothelial cells in vivo (Heuer et al., similar if not identical. Obviously, there is a family of struc- 1990; Peters et al., 1992). Our in situ hybridization data on turally related molecules synthesized by endothelial cells. embryonic, postnatal and adult tissue sections confirmed The question arises how many members this family may that tie-2 is specifically expressed in endothelial cells. The have? Dumont et al. (1992) detected several mRNA species spatial expression pattern of tie-2 was found to be identical by northern analysis in embryonic heart tissue hybridizing to the immunohistochemical staining for the endothelial cell with a tek probe. These findings can be explained by dif- marker PECAM(CD 31) (Newman et al., 1990). ferential, perhaps organ-specific splice events that could The developing brain does not contain endogenous endo- lead to the production of truncated and/or cytoplasmic thelial cell precursors (Noden, 1990). As a consequence, this tyrosine kinases. Alternatively, by using a cDNA encoding organ is vascularized by angiogenic sprouting from the the highly conserved catalytic domain as a probe, they may meningeal vascular plexus (Bär, 1980). The whole process have detected closely related molecules. This possibility consists of a complex pattern of physiological events cannot be excluded, because we used the less conserved part involving sprouting, migration and proliferation of endo- of the tie-2 cDNA and detected only one single mRNA thelial cells and finally their differentiation into tube-like species. It will therefore be interesting to see whether this structures surrounded by a basal lamina. Due to its still small family will grow by additional members. expression pattern, tie-2 could be involved in any of this The fact that we could detect tie-2 mRNA in a capillary events. tie-2 synthesis in the vascular plexus could reflect fraction highly enriched for endothelial cells gave us the first the competence of endothelial cells to respond to an angio- hint for an endothelium-specific expression pattern of this genic stimulus. Expression both at the tip of an invading gene. This finding confirmed the suitability of our experi- capillary and in more advanced vessels would argue for a mental design. Instead of cultured endothelial cells, we used participation in migration and proliferation. In the adult freshly prepared capillary material from P4 mouse brain as brain, tie-2 seemed to be down regulated and was detectable a mRNA source for the initial cloning step. There were two mainly in larger vessels of the meninges. Because endo- reasons for this approach. First, the proliferation of capil- thelial cell turnover is low in the adult, persistent expression laries is maximal during the first postnatal days in the rodent 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 Fig. 4. tie-2 expression during brain development. Sagittal changes during embryonic development (Risau and sections of adult brain (A,B), postnatal day 4 brain (C,D,G,H) and Lemmon, 1988), tie-2 with its peculiar organization of extra- embryonic day 12.5 brain (E,F) were hybridized with a tie-2 cellular domains may be involved in the regulation of endo- probe. Bright-field (A,C,E) and corresponding dark-field thelial cell-substratum adhesion. micrographs (B,D,F) Arrowheads indicate expression in It is unlikely that the multiple aspects of endothelial cell capillaries and arrows indicate hybridization signals over physiology are controlled by a single receptor. Recently, we meningeal blood vessels. Higher magnification of a capillary have identified flk-1 as an endothelium-specific receptor sprout at the dorsal surface of a postnatal day 4 telencephalon (G). Higher magnification of a capillary in a deeper layer of the brain tyrosine kinase in an identical approach as described here (H). No other than the vascular elements of the brain were found for tie-2 (Millauer et al., 1993). The temporal and spatial to synthesize tie-2 mRNA. LM, leptomeninges; LV, lateral expression pattern of flk-1, which is a receptor for vascular ventricle; TE, telencephalon; (A, B, C, D, E, F) bar represents 110 endothelial cell growth factor (VEGF), seems to be similar, mm; (G, H) bar represents 10 mm; if not identical to that found for tie-2. However, both 964 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 mm. receptors differ in topology, especially in their extracellular From transplantation studies in the quail-chick system, it domains. It is conceivable that a set of receptors, whose is well documented that the ability of endothelial cells to composition changes in a time- and tissue-dependent form a blood vessel system is not due to an intrinsic genetic manner, speciﬁes endothelial cell behaviour during blood program, but depends on environmental cues from the tissue vessel development and maintenance. to be vascularized (Noden, 1988; Stewart and Wiley, 1981; Ekblom et al., 1982). Given the ﬁnal morphological hetero- Fig. 6. tie-2 expression in organs and tissues of E 12.5 embryos geneity of the vasculature, it is clear that recognizing and and colocalization with immunostaining for PECAM (CD31). responding to different external signals must be a central Adjacent sections of E12.5 embryos were stained with an aspect in endothelial cell biology. The tie-2 receptor is ubiq- antibody recognizing PECAM(CD31) (A,C,E,G) or hybridized uitously expressed in the endothelium of every organ and 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). tissue that we have examined. Apart from brain, it was TE, telencephalon; LV, lateral ventricle; CP, choroid plexus of the detected in heart, lung, kidney, liver and spinal cord. It is lateral ventricle; DA, dorsal aorta; SC, spinal cord; EN, therefore likely that tie-2 is involved in a very fundamental endocardium; MY, myocardium; BR, bronchus; SO, somite; Bar signalling process, typical for the whole population of het- represents 110 mm. erogeneous endothelial cells. 966 H. Schnürch and W. Risau

The finding that tie-2 is expressed in E8.5 embryos (Coffman et al., 1990), vertebrate homologues of the confirms this notion and suggests a role even at the early D r o s o p h i l a n o t c h gene product. Tan-1 and Xotch are phases of vascular development. Blood islands are meso- integral membrane proteins, each containing 36 EGF-like dermal derivatives of the yolk sac with characteristic mor- repeats in the extracellular part. The molecules are thought phology. The appearance of a central cell cluster distinct to be involved in some sort of extracellular interaction. The from the peripheral cell layer, is the first overt sign for the A chain of laminin, a glycosylated basal lamina protein, separation of the hematopoietic and endothelial cell lineage. possesses 15 EGF-like repeats, three of them having a 30% We could detect tie-2 mRNA in the peripheral endothelial sequence identity with the EGF-like triplet of tie-2 (Sasaki cell precursors indicating that the gene could be involved in et al., 1988). Unlike TAN-1, Xotch and Laminin, the EGF- the onset of angioblast differentiation. tie-2 expression was like units in tie-2 (also tie and TEK) have eight instead of also observed in yolk sac mesoderm outside of blood six cysteine residues. It is an open question whether these islands. The significance of this observation is not clear. additional residues are also involved in disulphide bonding However, one may speculate that tie-2 mRNA synthesis and whether this has functional importance for the interac- reflects the segregation of endothelial cells and hematopoi- tion of tie-2 to its yet unidentified ligand. In any event, to etic cells on a molecular level before this process is mor- our knowledge tie and tie-2 are so far the only receptor phologically evident. tyrosine kinases known to have EGF-like repeats in their The origin and the differentiation of endothelial cells extracellular parts. within the embryo proper is less well understood. In contrast to EGF-like repeats, immunoglobulin-like However, from the transplantation studies mentioned domains are structural motifs present in the extracellular above, it is clear that endogenous endothelial cell precur- portions of wide variety of receptor tyrosine kinases (Ullrich sors are present in most mesodermal tissues of the embryo. and Schlessinger, 1990; Williams and Barclay, 1988). tie-2 The development of some intraembryonic blood vessels, has a single, amino-terminal Ig domain with the character- e.g. the dorsal aortae, has been described as an assembly istics of a C2-set sequence. In general, the function of Ig- of in situ differentiating endothelial cells (Meier, 1980; containing proteins can be described as mediating het- Pardanaud et al., 1987; Coffin and Poole, 1988; Pardanaud erophilic and homophilic binding events. In the case of the et al., 1989). This mode of blood vessel formation, which alpha-PDGF receptor, the three amino-terminal Ig domains is called vasculogenesis, is mechanistically distinct from determine the binding specificity for the PDGF AA ligand angiogenic sprouting (Pardanaud et al., 1987, 1989; Risau (Heidaran et al., 1990). et al., 1988). We found tie-2 mRNA expressed in cells Fibronectin type III (FN III) repeats are also present in assembled like a ring in the anlagen of the dorsal aortae. the extracellular parts of receptor tyrosine kinases The same pattern was found in the primordia of the including the insulin receptor, IGF-1 receptor (Ullrich et cardinal veins. Furthermore, tie-2 mRNA was present in al., 1986), sevenless (Norton et al., 1990), Eph (Hirai et the developing endocardium of the heart and in scattered al., 1987), cek5 (Pasquale, 1991), axl (O’Bryan et al., mesenchymal cells, which were not associated with a par- 1991) and ark (Rescigno et al., 1991). Originally described ticular structure. This expression profile could be indica- as internal homologous units in the extracellular matrix tive of the presence of intraembryonic endothelial cell pre- (ECM) protein fibronectin (Petersen et al., 1983), FN III cursors before and at the time of differentiation. In that repeats have been identified in, for example, adhesion case, tie-2 would be an early marker for intra- and extraem- molecules, cytokine receptors and collagens. In the tenth bryonic angioblasts and molecular probes for tie-2 would FN III repeat of fibronectin, the tripeptide RGD was found be a valuable tool for studying the early events of vascular to be crucial for the interaction of the ECM protein with d e v e l o p m e n t . integrins on the cell surface (Ruoslahti and Pierschbacher, Endothelial cells are able to interpret external cues for 1986). The three FN III repeats of tie-2 are mostly related their correct assembly into a highly diverse blood vessel to those found in protein-tyrosine phosphatases and in the system. The tie-2 receptor may be considered as part of a axonal glycoprotein TAG-1. A possible RGD cell attach- signaling system allowing endothelial cells to respond to ment site is present between the amino-terminal Ig domain soluble or matrix-bound factors and to communicate with and the three EGF-like repeats of tie-2. The significance of other cells. The exact role of tie-2 on a molecular level has this observation is unclear, because the tripeptide sequence not been determined yet. The intracellular portion of the is neither in a FN III repeat context nor is it conserved in deduced protein contains a typical tyrosine kinase domain. tie and TEK. Interestingly, the ECM protein laminin The identification of substrates for phosphorylation and the appears to mediate its effects on endothelial cells in part question whether there exists a endothelial cell-specific by a RGD-containing sequence in the A chain (Grant et al., signaling transduction pathway will be an area of future 1 9 8 9 ) . research. In the extracellular portion of tie-2 is a mosaic of Taken together, tie-2 has significant similarities to ECM immunoglobulin, EGF-like and fibronectin type III proteins and adhesion molecules. Whether these homologies domains. EGF-like repeats are present in numerous, appar- indicate a role for this receptor tyrosine kinase in cell-cell ently unrelated proteins. The functional significance of this or cell-matrix interactions, or whether the ligand for tie-2 is sequence motif is not obvious. Several copies can be a diffusible molecule remains to be determined. Due to the present in the extracellular part of membrane-bound unusual topology of tie-2, the ligand could be a novel proteins or in polypeptides that are secreted. The three molecule whose identification could lead to a more complete EGF-like repeats in tie-2 show about 30% sequence simi- understanding of the molecular mechanisms of blood vessel larity with TAN-1 (Ellisen et al., 1991) and Xotch formation. tie-2 expression during vascular development 967

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