Present address: Department of Developmental Neurobiology, NIMR, The RIdgeway, Mill Hill, London NW7 1AA, UK
1 Division of Biology 216-76, California Institute of Technology, Pasadena, CA 91125, USA
2 Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA
*Author for correspondence (e-mail: c/o mancusog{at}cco.caltech.edu)
Accepted 19 December 2001
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SUMMARY |
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Key words: EphB4, EphrinB2, Angiogenesis, Vasculature, Mouse
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INTRODUCTION |
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Angiogenesis involves a complex series of reciprocal interactions between the endothelial cells of the developing blood vessels and neighboring cells. Perivascular mesenchymal cells provide endothelial cells with signals such as vascular endothelial growth factor (VEGF) (Carmeliet et al., 1996; Ferrara et al., 1996
) and angiopoietin 1 (Ang1) (Davis et al., 1996
; Suri et al., 1996
; Sato et al., 1995
; Dumont et al., 1994
). In turn, endothelial cells send signals of their own, such as platelet-derived growth factor (PDGF) (Hellstrom et al., 1999
; Hirschi et al., 1999
), transforming growth factor ß (TGFß) (Goumans et al., 1999
; Pepper, 1997
; Oshima et al., 1996
; Dickson et al., 1995
) and neuregulin (Kramer et al., 1996
; Meyer and Birchmeier, 1995
; Marchionni et al., 1993
), to surrounding support cells (reviewed by Flamme et al., 1997
; Hanahan, 1997
). Endothelial cells must also interact with one another, coordinating vessel integrity, identity, growth and remodeling via such molecules as Delta-4 (Krebs et al., 2000
; Shutter et al., 2000
), integrin avb3 (Friedlander et al., 1995
; Brooks et al., 1994
) and VE-cadherin (Gory-Faure et al., 1999
; Breier et al., 1996
). Therefore, both endothelial-support cell and inter-endothelial communication are required for proper vessel assembly (Gale and Yancopoulos, 1999
; Hanahan, 1997
; Folkman and DAmore, 1996
; Risau and Flamme, 1995
).
EPH receptor tyrosine kinases and their transmembrane ligands, the ephrins (Wilkinson, 2000), have recently been shown to be expressed in and around the developing circulatory system (reviewed by Adams and Klein, 2000
). Several studies have implicated ephrin signaling in endothelial cell behavior and angiogenesis (Adams et al., 2001
; Helbling et al., 2000
; Adams et al., 1999
; Gerety et al., 1999
; Stein et al., 1998
; Wang et al., 1998
; Daniel et al., 1996
; Stein et al., 1996
). EphrinB2 (Efnb2 Mouse Genome Informatics) is specifically expressed in arterial endothelial cells, and ephrinB2 homozygous mutant mice die at E9.5 with severe cardiovascular defects (Adams et al., 1999
; Wang et al., 1998
). To date, ephrinB2 is the only ephrin ligand whose knockout shows an angiogenic phenotype (Adams et al., 1999
). Deletion of the ephrinB2 cytoplasmic domain yields an identical vascular phenotype, consistent with the notion that this transmembrane ligand functions in reverse signaling (Adams et al., 2001
).
Initial studies suggested that the main ligand function of ephrinB2 is exerted in the arterial endothelium, where it mediates signaling to veins via EphB4 (Ephb4 Mouse Genome Informatics), a receptor more abundantly expressed on venous endothelial cells (Gerety et al., 1999; Wang et al., 1998
). Consistent with this notion, Ephb4 mutant mice show angiogenic defects similar or identical to those in ephrinB2/ mice (Gerety et al., 1999
). However, in contrast to Ephb4, which is restricted to the cardiovascular system, expression of ephrinB2 is not restricted to endothelial cells, but is also found in mesenchymal cells, pericytes and vascular smooth muscle cells surrounding sites of active angiogenesis (Gale et al., 2001
; Shin et al., 2001
; Adams et al., 1999
; Wang et al., 1998
). These observations suggested that ephrinB2 might mediate support cell to endothelial cell communication, as well as inter-endothelial interactions (Adams et al., 1999
; Gale and Yancopoulos, 1999
; Gerety et al., 1999
). Consistent with this, recent studies in mouse (Adams et al., 1999
) and Xenopus (Helbling et al., 2000
) have attributed a role to mesenchyme-derived ephrinB2 signals in restricting blood vessel growth to the intersomitic space. In these experiments, however, ephrin signaling was disrupted throughout the embryo, obscuring its essential site of action.
We were interested in determining whether endothelial ephrinB2 expression is essential for angiogenesis, or if ephrinB2 derived from perivascular mesenchyme is sufficient to drive vascular remodeling. We therefore constructed a conditional allele of ephrinB2 that can be excised by the expression of Cre recombinase (Cre) in a tissue of interest (Nagy, 2000; Orban et al., 1992
). Endothelial-specific deletion was accomplished by the use of a transgenic Tie2-Cre mouse line that expresses Cre in endothelial cells of the embryo (Kisanuki et al., 2001
). This endothelial specific knockout of ephrinB2 leads to angiogenic remodeling and cardiac defects that are indistinguishable from those of the conventional ephrinB2 knockout (Wang et al., 1998
). These data demonstrate that ephrinB2 is absolutely required in the endothelial and endocardial cells of the developing mouse embryo for proper cardiovascular development of both arteries and veins. In all cases examined, ephrinB2 expression in adjacent mesenchymal tissue was not sufficient to compensate for the loss of endothelial expression in neighboring vessels.
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MATERIALS AND METHODS |
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To remove the Floxed PGKneo selection cassette, we used transient expression of Cre recombinase. Uncut pBS185 plasmid (Invitrogen) containing CMV-driven Cre recombinase expression was electroporated into homologously recombinant ES cells, and the cells were plated at high density. After 48 hours of growth, the cells were trypsinized and replated at low density. Between 10 and 14 days of growth, individual colonies were picked and replated into 96-well plates. Southern blot analyses of genomic DNA isolated from plate replicates identified ES clones that had undergone deletion of the PGKneo cassette but retained an intact exon I (Fig. 1A, Floxed locus; Fig. 1C, Neo deleted). Genomic DNA was cut with HindIII, and a 1.2 kb EcoRI-HindIII genomic fragment was used as a Southern probe (Fig. 1A, Probe B). Wild-type, exon 1-deleted and fully deleted loci were indistinguishable using this Southern blot strategy, as they all generate a band of approximately 6kb (Fig. 1C, Wildtype). Blastocyst injection of floxed ephrinB2 ES cells were performed essentially as described (Ma et al., 1998). Germline chimeras were crossed onto a pure C57/Bl6 background and all subsequent breeding was done in a C57/Bl6 background.
Genotyping
PCR genotyping for the conditional ephrinB2 allele (floxed allele) was performed with a 5' primer specific for the 5' loxP site insertion, 5'-AAGTTATAAGCTTCAACGCGTCC-3' (TF3), and a 3' primer in the genomic region downstream of exon 1, 5'-GAGCCCCAGGTTCTAGAATAACTTCG-3' (RF1) (product size of 320 bp). Genotyping of the ephrinB2-lacZ allele was carried out with the following lacZ-specific primer pair: 5'-CGCCCGTTGCACCACAGATG-3' (UX-161) and 5'-CCAGCTGGCGTAATAGCGAAG-3' (UX-160G) (product size of 370 bp). The Tie2-Cre transgene was detected by allele specific primers, with a 5' primer in the Tie2 promoter, 5'-GGGAAGTCGCAAAGTTGTGAGTT-3' (Tie2T5F1) and a 3' primer in the Cre gene, 5'-CTAGAGCCTGTTTTGCACGTTC-3' (Cre2) (product size of 490 bp). The wild-type ephrinB2 locus was detected with a 5' primer that includes sequence flanking the inserted 5' loxP site, and a 3' primer downstream of the first exon, 5'-GCTGCCCGCGGCCGGTCCCAACG-3' (BrgF1) and 5'-CCGTTAGTGGCAACGTCCTCCGTCCTCG-3' (HL-I-R2h) (product size of 580bp). Conditional knockout mice were identified by the presence of loxP-allele specific, lacZ allele specific and Tie2-Cre specific PCR products. Homozygous ephrinB2-lacZ embryos were identified by the presence of lacZ-specific PCR products and the absence of wild-type ephrinB2-specific PCR products (in duplicate). Homozygous ephrinB2-loxP mice were identified by the presence of loxP-allele specific PCR products and the absence of wild-type ephrinB2-specific PCR products. In all embryos, a small amount of tissue was collected from the tail region for genomic DNA isolation and genotyping. To demonstrate deletion of ephrinB2-loxP exon I in vivo, the following primers were used to distinguish intact (636 bp) and deleted (309 bp) ephrinB2-loxP alleles: 5'-CGGCCGGTCCATAACTTCGTATAGCA-3' (HLF1) and 5'-CCGTTAGTGGCAACGTCCTCCGTCCTCG-3' (HL-I-R2h).
To generate conditionally deleted ephrinB2 embryos, we first generated mice heterozygous for both the ephrinB2-lacZ allele and the Tie2-Cre transgene (ephrinB2lacZ/+;Tie2-Cre+). These mice were then crossed to ephrinB2-loxP (ephrinB2loxP/+) heterozygous (or ephrinB2loxP/loxP homozygous) mice. EphrinB2 conventional knockouts were generated by intercross of ephrinB2lacZ/+ heterozygotes (Wang et al., 1998). For vascular-specific Cre Recombinase expression, we used the Tie2-Cre transgenic mouse (Kisanuki et al., 2001
). The Tie2-Cre expression pattern was examined by crossing Tie2-Cre mice to the R26R Rosa lacZ reporter mice (Soriano, 1999
). Embryos were collected at E8.25-E9.5 and processed as described below by X-gal development or immunofluorescent double-labeling.
lacZ and immunohistochemical staining
To examine lacZ expression, mouse embryos were dissected between E8.25 and E9.5, fixed in 0.25% glutaraldehyde/PBS for 5 minutes, rinsed twice with PBS, and stained overnight at 37°C in X-Gal buffer [1.3 mg/ml potassium ferrocyanide, 1 mg/ml potassium ferricyanide, 0.2% Triton X-100, 1 mM MgCl2 and 1 mg/ml X-Gal in phosphate-buffered saline (PBS, pH 7.2)]. For antibody staining, embryos were first fixed overnight in 4% paraformaldehyde/PBS at 4°C. For section staining, embryos were embedded in 15% sucrose and 7.5% gelatin in PBS, and 15 µm sections were collected on a cryostat. Whole-mount staining procedures with anti-PECAM1 antibody (clone MEC 13.3, Pharmingen, 1:200 overnight at 4°C) and anti-ß-galactosidase antibody (3-prime 5-prime, 1:1000, overnight at 4°C) were essentially as described (Wang et al., 1998). Either HRP-conjugated secondary antibodies (Jackson, 1:200, overnight at 4°C) or secondary antibodies conjugated to FITC or Alexa-568 (Jackson, 1:200, and Molecular Probes 1:250, 1 hour at room temperature) were used for whole-mount staining. For immunofluorescent detection on sections, secondary antibodies conjugated to FITC or Alexa-568 (Jackson, 1:200, and Molecular Probes 1:250) were applied for 1 hour at room temperature. For whole-mount immunofluorescent staining, embryos were cleared in Vectorshield (Molecular Probes) for 20 minutes subsequent to the final antibody wash, then mounted on a slide whose coverslip was elevated by a bridge of two coverslips on each side. This enabled us to avoid crushing the embryos. All confocal microscopy was carried out on a Leica SP confocal (Leica). All brightfield images were captured using an Axiocam CCD camera (Zeiss).
In situ hybridization
In situ hybridization was carried out essentially as described (Wang et al., 1998; Birren et al., 1993
). E9 embryos were cryosectioned at 15 µm, and adjacent sections were hybridized with RNA probes against the ephrinB2 EC domain (Wang et al., 1998
) and Flk1 (Kdr Mouse Genome Informatics; a generous gift from T. Sato).
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RESULTS |
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To verify that the floxed ephrinB2 allele was able to undergo Cre-mediated deletion, ephrinB2loxP/+ mice were crossed to a CMV-Cre mouse, in which Cre is expressed in all cells of the early embryo (Zinyk et al., 1998). Embryos that inherited both the ephrinB2loxP allele and the CMV-Cre transgene show deletion of the loxP allele by PCR analysis (Fig. 1D, right panel, lower band, deleted). Embryos that inherited the ephrinB2loxP allele but no Cre transgene showed no deletion (Fig. 1D, left panel, upper band, intact). Sequencing of these PCR products confirmed the deletion event (data not shown). Deletion of ephrinB2 exon I by Cre recombinase removes the signal peptide, creating a null allele similar in structure to the original conventional ephrinB2 knockout allele (Wang et al., 1998
), but with no lacZ marker included.
Embryonic endothelial-specific Cre expression
To knock out ephrinB2 specifically in endothelial cells, we used an endothelial-specific Cre-expressing transgenic mouse line, Tie2-Cre (Kisanuki et al., 2001). Tie2, a pan-endothelial receptor tyrosine kinase, is expressed from the earliest timepoints of vascular development (Dumont et al., 1995
; Dumont et al., 1992
). The Tie2 promoter/enhancer is well characterized and has been shown to drive specifically transcription in the majority of embryonic endothelial cells (Schlaeger et al., 1995
; Schlaeger et al., 1997
).
ephrinB2 expression in the vasculature is first seen around E8.25, immediately after the formation of the primary vascular plexus (Wang et al., 1998). Homozygous ephrinB2lacZ/lacZ mutants first exhibit cardiovascular defects around E9 (Wang et al., 1998
). These two timepoints define the interval during which ephrinB2 function is first required in the vasculature. To verify that the Tie2-Cre transgene is expressed during this interval, we crossed Tie2-Cre+ mice to the Rosa26 reporter (R26R) strain (Soriano, 1999
). In the R26R reporter mice, a floxed transcriptional/translational stop cassette (floxed STOP) (Lakso et al., 1992
) is present between the ubiquitously expressing Rosa26 promoter and the lacZ gene. Any cell expressing Cre will excise the floxed STOP, allowing lacZ expression in that cell and all its progeny (Nagy, 2000
). Using this reporter line, we confirmed that Tie2-Cre is active throughout the vasculature as early as E8.25 (Fig. 2). At this stage, the yolk sac, a highly vascularized extra-embryonic tissue, shows widespread Tie2-Cre activity (Fig. 2A YS). The primitive vasculature of the embryo proper also shows Tie2-Cre activity (Fig. 2A, arrowheads) throughout the vessels of the head, heart region and trunk (Fig. 2C-E, respectively, arrowheads). The endothelial lining of the heart, the endocardium, is also positive, as expected (Fig. 2E, arrows) (Kisanuki et al., 2001
; Schlaeger et al., 1997
). As ephrinB2 activity is thought to play a role in angiogenic sprouting (Adams et al., 1999
), we wanted to confirm that Tie2-Cre was active in endothelial sprouts. Intersomitic vessels derived from vascular sprouts of the dorsal aorta showed clear Tie2-Cre activity (Fig. 2B, arrows, DA). Thus, Tie2-Cre is specifically expressed in the endothelium, including angiogenic sprouts, from a timepoint early enough to delete ephrinB2 when its angiogenic function is first required (Adams et al., 1999
; Wang et al., 1998
).
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EphrinB2 expressed in arteries is required for remodeling of the anterior cardinal vein
The anterior cardinal veins are the main vessels that transport blood from the head back through the sinus venosus to the heart. These lateral vessels appear around E8.5, after the formation of the dorsal aorta. Each ACV arises initially from the fusion of multiple small vessels present in the lateral mesenchyme of the hindbrain and head (Coffin and Poole, 1988). The early, small diameter vessels that will give rise to the ACVs express EphB4 receptor (Gerety et al., 1999
). EphrinB2 is expressed extensively in the hindbrain mesenchyme surrounding the developing ACV, as well as in neighboring neuroepithelium (Fig. 5A-C, red channel, black and white arrowheads, respectively) (Wang et al., 1998
). Both ephrinB2lacZ and Ephb4lacZ homozygous embryos show a failure of ACV assembly, resulting in a plexus of disorganized small-diameter vessels (Fig. 5N) (Adams et al., 1999
; Gerety et al., 1999
). This angiogenic remodeling defect, in a place where no arteriovenous (AV) interface is apparent at E9.5, suggested that ephrinB2 from perivascular mesenchymal cells might signal to EphB4-expressing vessels (Gerety et al., 1999
). Double-labeling of E9.5 embryos from a Tie2-Cre X R26R lacZ reporter cross with anti-PECAM1 and anti-ß-Gal confirmed that Tie2-Cre is active only in the endothelium of the hindbrain (Fig. 5D-F, green and red channels, respectively, arrows). We confirmed vessel-specific deletion by in situ hybridization on conventionally and conditionally knocked-out embryos and littermates (Fig. 5G-L). Comparison of ephrinB2 probe and Flk1 probe staining shows that ephrinB2 was selectively lost in the endothelium (Fig. 5I,L versus 5G,J, arrows), and was still present in the non-vascular tissues (Fig. 5G versus I, arrowheads) of conditional knockout embryos. Flk1 in situ hybridization signals confirmed the presence of endothelial cells in these samples (Fig. 5J-L).
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Endothelial ephrinB2 is required for angiogenesis of arteries in the head
Vascularization of the head results in a characteristic hierarchical branching pattern of large to small vessels, including morphogenesis of the internal carotid artery (ICA) (Coffin and Poole, 1988). In ephrinB2lacZ/lacZ homozygous mutant embryos, this network does not develop properly, resulting in a disorganized, often fused network of capillaries (Fig. 6N) (Adams et al., 1999
; Wang et al., 1998
). EphrinB2 is expressed in arterial endothelial cells of the head (Fig. 6A,B, red channel, arrows), as well as extensively in the mesenchyme and neuroepithelium of the developing brain (Fig. 6A,B, red channel, black and white arrowheads respectively) (Wang et al., 1998
). Previous studies have indicted that ephrinB2 function is essential for angiogenesis of vessels in the head, but were unable to distinguish an autonomous requirement for ephrinB2 in the blood vessels, from a requirement in the neighboring head mesenchyme or neuroepithelium (Adams et al., 1999
; Wang et al., 1998
).
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In ephrinB2lacZ/loxP;Tie2-Cre+ embryos the head vasculature fails to assemble correctly (Fig. 6M versus 6O). The initial head plexus forms, but subsequently remains in a primitive state, a disorganized network of dilated capillaries. The ephrinB2lacZ/lacZ homozygous mutant embryos exhibit an identical phenotype (compare Fig. 6N versus 6O). Thus, in the conditional knockout of ephrinB2, the absence of endothelial ephrinB2 expression results in defective remodeling of the ICA, as well as the branches of the ACV, despite high mesenchymal and neuroepithelial ephrinB2 expression at this stage (Fig. 6A,B, red channel, arrowheads). These data indicate that endothelial ephrinB2 is required autonomously in arteries for proper arterial angiogenesis.
To determine whether the angiogenic phenotype of the conditional mutant reflects aberrant perivascular smooth muscle cell (SMC) recruitment or differentiation, we stained embryos at E9.5 with antibody to smooth muscle actin (
SMA). At this stage, however, there was not yet any
SMA expression in wild-type embryos in the smaller peripheral vessels of the ICA where phenotypic defects are observed in the mutant (data not shown). Therefore, it seems unlikely that a defect in SMC differentiation can account for the defective angiogenesis observed in the mutants. However, it is possible that earlier markers of undifferentiated pericytes might reveal such a defect.
Endothelial ephrinB2 is required for angiogenesis of intersomitic vessels
The embryonic trunk is initially vascularized by a series of intersomitic vessels (ISVs) that arise from branches of the dorsal aorta (DA) and posterior cardinal veins. These vessels grow between adjacent somites to form a simple interconnected network dorsally around E8.75. Through extensive angiogenesis, this primitive structure elaborates an intricate network of small capillaries (Fig. 7M), some of which eventually invade the developing neural tube and flanking somites (Drake and Fleming, 2000; Coffin and Poole, 1988
).
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To confirm that Cre activity is restricted to ISVs in the trunk, we examined embryos from a Tie2-Cre X R26R lacZ reporter intercross. Double-staining of such embryos for PECAM1 and ß-gal confirmed that the lacZ reporter was specifically activated in the vessels (Fig. 7D-F, arrows), and not in the somites or other surrounding tissues (compare Fig. 7A with 7D). These data suggested that ephrinB2 expression should be selectively eliminated in the ISVs of ephrinB2lacZ/loxP;Tie2-Cre+ mice. To confirm this, in situ hybridization with ephrinB2 and Flk1 RNA probes was performed. Consistent with the Tie2-Cre X reporter data (Fig. 7D-F), these experiments indicated that in the conditional knockout, ephrinB2 is selectively lost in the vasculature (Fig. 7G-I versus J-L, insets, arrows, V), but is still present in somites (Fig. 7G-I, arrowheads, and insets, S). Endothelial cells are still present, however, as revealed by Flk1 probe signals in all genotypes (Fig. 7J-L, arrows, and insets, V). Complete loss of ephrinB2 in situ signal in the ephrinB2 conventional knockout confirmed the specificity of the riboprobes used (Fig. 7H).
Endothelial-specific ephrinB2 knockout embryos (ephrinB2lacZ/loxP;Tie2-Cre+) show an arrest in intersomitic vessel angiogenesis at the primary plexus stage (Fig. 7O, compare with 7M). The vessels appear fused dorsally with little or no branching. This phenotype is identical to that of the ephrinB2lacZ/lacZ mice (compare Fig. 7N with 7O) (Gerety et al., 1999). These data indicate that ephrinB2 is required in the intersomitic arteries for proper angiogenesis to occur. Thus, remodeling of ISVs requires ephrinB2-EphB4-mediated interactions between ISVs. Somite-derived ephrinB2 signal is apparently not sufficient to compensate for the requirement for ephrinB2 in these vessels.
We did not observe aberrant branching of ISVs into somitic mesenchyme in either our conventional or conditional ephrinB2 knockouts (Fig. 7N,O, arrowheads). This is in contrast to the phenotype described by Adams et al., in their conventional ephrinB2 knockout (Adams et al., 1999), as well as in a study employing mis-expression of dominant-negative Ephb4 alleles in Xenopus (Helbling et al., 2000
), both of which describe aberrant ISV branches into adjacent somites. The difference in the penetrance of the ISV branching phenotype between the two conventional ephrinB2 mutations may reflect differences in genetic background (Gupta et al., 2001
; Rohan et al., 2000
). Consequently, we were unable to determine whether arterial ephrinB2 expression is required for proper intersomitic guidance of the ISVs. The question of whether ephrinB2 in somitic mesenchyme plays a role in guidance of ISVs will require a conditional knockout of the gene specifically in that tissue, on a genetic background that allows the penetrance of that phenotype.
Endocardial ephrinB2 is required for heart development
The endothelial lining of the early embryonic heart, the endocardium, is similar in many respects to the rest of the vasculature, in terms of gene expression, and cell behavior (reviewed by Gale and Yancopoulos, 1999; Brutsaert et al., 1998
; Dumont et al., 1992
; Dumont et al., 1995
). Subsequent interactions with its specialized tissue environment leads to morphological changes, including heart looping and myocardial trabeculation, the formation of endothelial-cell lined projections from the supporting myocardium (Fishman and Chien, 1997
). In the heart, ephrinB2 is expressed primarily in the endocardium (Fig. 8A-C, red channel, arrows) (Wang et al., 1998
). EphrinB2 is also weakly expressed in myocardium or other support cells (Fig. 8A-C, red channel, arrowheads) (Wang et al., 1998
). The heart phenotype in the conventional ephrinB2 knockout is an arrest of development resulting in no looping and little or no myocardial trabeculation, and frequent abnormal swelling of the heart (Adams et al., 1999
; Gerety et al., 1999
; Wang et al., 1998
).
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DISCUSSION |
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The functional requirement for ephrinB2 is intrinsic to the cardiovascular system
Our data demonstrate that ephrinB2 is required in arterial endothelium for the remodeling of veins of the head, ACV and yolk sac, most probably by signaling through EphB4 receptor in these vessels. This ligand appears to be required cell-autonomously in arteries as well, without which angiogenic remodeling of the ICA and vitelline artery is disrupted in the head and yolk sac, respectively. Although ephrinB2 is expressed at high levels in the somites flanking the developing ISVs, endothelial ephrinB2 is still required for the elaboration of a fine capillary network from the primitive intersomitic arteries and veins. These data, therefore, appear to reveal instances of forward, reverse and bi-directional signaling between arterial and venous vessels. Our results also demonstrate that even at sites of high mesenchymal ephrinB2, vascular expression is still absolutely essential, and argue that mesenchymal ephrinB2 alone is unable to support angiogenesis.
Our data do not exclude the possibility, however, that somitic ephrinB2 contributes to ISV growth and guidance. Adams et al., observe ISVs branching aberrantly into the somites in their ephrinB2 knockout, suggesting that ephrinB2 in the somites plays repulsive role, restricting the growth of ISVs to the intersomitic space (Adams et al., 1999). Our ephrinB2 knockout mice do not show a similar aberrant branching phenotype (Gerety et al., 1999
). The reason for this difference is not clear. Strain differences may account for this discrepancy (Gupta et al., 2001
; Rohan et al., 2000
). Consistent with this, Adams et al. find ephrinB2 heterozygous offspring at half the expected proportions (Adams et al., 1999
), while our ephrinB2 offspring are found at Mendelian ratios, suggestive of a reduced penetrance of the ephrinB2 mutant phenotype. Forced expression of ephrins or dominant-negative EphB4 receptor throughout the developing Xenopus embryo has a similar effect on ISV growth (Helbling et al., 2000
). This ISV guidance model fits well with the repulsive guidance role ascribed to ephrinB2 signaling in neural crest migration and axon pathfinding in and around somitic tissue (Krull et al., 1997
; Wang and Anderson, 1997
). However, somite-specific deletion of ephrinB2 will be required to confirm that it exerts this ISV guidance function from non-vascular tissue.
Necessity versus sufficiency: does mesenchymal ephrinB2 have a role in angiogenesis?
Our loss-of-function results provide evidence of the necessity of endothelial ephrinB2 in angiogenesis, and indicate that mesenchymal ephrinB2 is insufficient to compensate for its loss from vessels. However, this does not address whether mesenchymal ephrinB2 expression is also required for angiogenesis. Given the extensive expression of ephrinB2 in the mesenchyme surrounding vessels in the head, trunk (Adams et al., 1999; Wang et al., 1998
) and in smooth muscle (Gale et al., 2001
; Shin et al., 2001
), it is possible that this non-endothelial expression is required for angiogenesis in parallel with its requirement in the endothelium. If so, then the fact that the phenotypes of the conventional and endothelial-specific ephrinB2 knockouts are identical argues that such a parallel function for ephrinB2 in endothelial and mesenchymal cells must be non-redundant. Alternatively, the presence of ephrinB2 in the early embryonic mesenchyme may be irrelevant to angiogenesis, but instead reflects other potential roles, such as somite patterning (Durbin et al., 1998
), neural crest migration (Adams et al., 2001
; Krull et al., 1997
; Wang and Anderson, 1997
), hindbrain segmentation (Xu et al., 1995
; Xu et al., 1996
) and axon guidance (Frisen et al., 1998
; Drescher et al., 1995
). The direct test of a mesenchymal requirement for ephrinB2 in angiogenesis awaits the identification of mesenchymal promoter elements with which to generate mesenchyme-specific Cre deleter mice.
ACV remodeling requires artery to vein ephrin signaling
It has been hypothesized that the failure of ACV primordium to remodel into single-vessel structures in the ephrinB2 and Ephb4 mutants was the result of a loss of ephrinB2 stimulation from the adjacent mesenchyme (Adams et al., 1999; Gerety et al., 1999
). An important factor in that interpretation was the lack of obvious AV interface between this venous structure and any arterial ephrinB2-expressing vessels. Based on this, we expected that in an endothelial-specific knockout of ephrinB2, we would see rescue of the ACV phenotype. Surprisingly, the ACV phenotype of our conditional knockout is identical to that of the conventional ephrinB2 knockout (Adams et al., 1999
; Gerety et al., 1999
). This suggests that an arterial source of ephrinB2 is required for ACV morphogenesis, such as the dorsal aorta. How could a physically remote tissue send a signal that is by nature membrane bound and requires cell-cell contact for transmission? Further analysis revealed transient endothelial continuity between the dorsal aorta and the developing ACV plexus at developmentally relevant stages during the assembly of these vessels. Based on the combination of conditional knockout phenotypes and the dorsal aorta-ACV contacts present in young embryos, we believe that the development of the ACV may require transient artery-vein interactions. This suggests that ACV development proceeds in a fashion similar to the rest of the vasculature, through AV interactions during which some vessels undergo pruning. Alternately, defective angiogenesis in the ACV might instead be due to insufficient blood flow resulting from aberrant cardiac development and function (see next section).
Heart morphogenesis requires endocardial ephrinB2 expression
During embryonic heart morphogenesis, essential interactions take place between endocardial cells (Gory-Faure et al., 1999), and between endocardial and myocardial cells (Meyer and Birchmeier, 1995
) in a reciprocal manner (Carmeliet et al., 1996
; Suri et al., 1996
). These tissue relationships are essential for the remodeling of the primitive heart tube to the looped, highly trabeculated structure that emerges at E9.5 (Gale and Yancopoulos, 1999
). Ephrin/Eph signaling has been implicated in these morphogenetic events both by expression and mutant phenotypes (reviewed by Adams and Klein, 2000
). Because EphB4 is expressed in endocardial and not myocardial cells, the failure of myocardial trabeculation in the Ephb4 knockout demonstrates that Ephrin signals must be received by the endocardium (Gerety et al., 1999
). Establishing the required source for the ephrinB2 signal is complicated again by the presence of this ligand in both the endocardium and the myocardium (Wang et al., 1998
). Although the expression levels in the myocardium are much lower than in the endocardium, the possibility remained that the requisite Ephrin signal originates in the myocardium. We now show that endocardial ephrinB2 function is absolutely required for heart morphogenesis, and is not compensated for by myocardial ephrinB2. This indicates that ephrinB2-EphB4-mediated signaling between endocardial cells is required for this morphogenetic program to be executed.
The close temporal relationship between vascular and cardiac phenotypes in knockouts of most genes encoding angiogenic signaling molecules or their receptors (Gerety et al., 1999; Gory-Faure et al., 1999
; Asahara et al., 1998
; Carmeliet et al., 1996
; Ferrara et al., 1996
; Dickson et al., 1995
; Sato et al., 1995
; Dumont et al., 1994
) invariably complicates phenotypic analysis and interpretation: a defect in peripheral angiogenesis could be the result of defective cardiac development and aberrant blood flow; conversely, defective heart development could be due to an obstructed or disorganized vasculature. We do observe heartbeat and blood flow in some conditional ephrinB2 mutant embryos with vascular defects, arguing that the defective peripheral angiogenesis in such mutants is not simply due to a complete lack of blood flow. However, aberrant hemodynamics could still contribute to the peripheral angiogenic defects seen in mutant embryos. Resolution of this issue awaits the development of appropriate Cre deleter transgenic mouse lines to temporally bypass the early cardiac requirement for ephrinB2 function, or alternatively identification of endothelial- or endocardial-specific promoter elements (Fishman, 1997
), for loss-of-function or rescue experiments, respectively.
EphrinB1 does not compensate for loss of endothelial ephrinB2
EphrinB1 is co-expressed with ephrinB2 in arteries (Adams et al., 1999), but cannot compensate for the loss of ephrinB2 in a conventional knockout (Adams et al., 1999
; Wang et al., 1998
). The perivascular expression of these ligands, however, does not fully overlap (Wang and Anderson, 1997
). Previously, therefore, one could have argued that the failure of ephrinB1 to compensate for ephrinB2 in the conventional knockout might reflect a requirement for ephrinB2 function in tissues where ephrinB1 is not expressed. However, the present data indicate that ephrinB1 cannot compensate for ephrinB2 within the cardiovascular system. This failure may reflect crucial differences in expression levels between the two ligands (Stein et al., 1998
), or alternatively structural differences that create different functional properties. For example, ephrinB2 is the only ligand that can bind efficiently to EphB4 (Sakano et al., 1996
; Brambilla et al., 1995
). Although veins express other EphB receptors that can interact with ephrinB1, only EphB4 is essential for angiogenesis (Adams et al., 1999
; Gerety et al., 1999
). Finally, differences in expression patterns within the cardiovascular system could explain the inability of ephrinB1 to compensate for ephrinB2: although ephrinB2 is expressed only in arterial vessels (Adams et al., 1999
; Wang et al., 1998
), ephrinB1 is expressed in all vessels (Adams et al., 1999
). The arterial restriction of ephrinB2 may therefore be an important aspect of its role in angiogenesis. Gene swapping experiments should reveal whether differences in the expression or activity of ephrinB1 and ephrinB2 account for their functional distinction.
Reverse signaling by ephrinB2 in angiogenesis
The interpretation of the vascular defects in the original ephrinB2 knockout was that reciprocal signaling between arterial ephrinB2 and venous EphB4 is required for the remodeling of both arteries and veins (Wang et al., 1998). An essential feature of this model is that upon engaging EphB4 receptors on veins, ephrinB2 functions as a receptor in arteries. This idea is supported by studies demonstrating that ephrinB cytoplasmic domains can undergo phosphorylation upon receptor binding (reviewed by Adams et al., 2001
; Wilkinson, 2000
; Mellitzer et al., 1999
; Xu et al., 1999
; Bruckner et al., 1997
; Holland et al., 1996
). Furthermore, a knockout of the ephrinB2 intracellular domain shows that the cytoplasmic tail of ephrinB2 is required for vascular morphogenesis (Adams et al., 2001
). These data, and the fact that the Ephb4 mutation causes arterial as well as venous defects, suggest a requirement for reciprocal signaling by Eph receptors to ephrinB2 in vascular remodeling. Our results take this one step further, showing that, in fact, this reverse signal must be received by arterial endothelial cells and/or endocardial cells for angiogenesis to occur. Taken together, these data reinforce the idea that bi-directional signaling between ephrinB2 and EphB4 in the cardiovascular system is essential for angiogenesis (Wang et al., 1998
).
Recent publications have highlighted the fact that many ephrins and Eph receptors are expressed in and around the adult vasculature at sites of active angiogenesis such as wound-healing and tumor angiogenesis, both in mice (Gale et al., 2001; Shin et al., 2001
) and humans (reviewed by Takai et al., 2001
; Dodelet and Pasquale, 2000
; Ogawa et al., 2000
; Berclaz et al., 1996
). These reports hint at potential roles for ephrins and Ephs in normal and pathological angiogenesis in the adult. Establishing whether the adult expression patterns of these ligands and receptors reflect functional roles in these angiogenic events will be an important step in determining the potential relevance of ephrin/Eph targeting drugs for pro- or anti-angiogenic therapies of cardiovascular disease and cancer, respectively. Our study has demonstrated the potential of conditional knockouts in understanding ephrin function and expression, and provides a useful mouse model system to further examine these issues in the adult.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Adams, R. H. and Klein, R. (2000). Eph receptors and ephrin ligands. essential mediators of vascular development. Trends Cardiovasc. Med. 10, 183-188.[Medline]
Adams, R. H., Wilkinson, G. A., Weiss, C., Diella, F., Gale, N. W., Deutsch, U., Risau, W. and Klein, R. (1999). Roles of ephrinB ligands and EphB receptors in cardiovascular development: demarcation of arterial/ venous domains, vascular morphogenesis, and sprouting angiogenesis. Genes Dev. 13, 295-306.
Adams, R. H., Diella, F., Hennig, S., Helmbacher, F., Deutsch, U. and Klein, R. (2001). The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration. Cell 104, 57-69.[Medline]
Asahara, T., Chen, D., Takahashi, T., Fujikawa, K., Kearney, M., Magner, M., Yancopoulos, G. D. and Isner, J. M. (1998). Tie2 receptor ligands, Angiopoietin-1 and Angiopoietin-2 modulate VEGF-induced postnatal neovascularization. Circ. Res. 83, 233-240.
Berclaz, G., Andres, A. C., Albrecht, D., Dreher, E., Ziemiecki, A., Gusterson, B. A. and Crompton, M. R. (1996). Expression of the receptor protein tyrosine kinase myk-1/htk in normal and malignant mammary epithelium. Biochem. Biophys. Res. Commun. 226, 869-875.[Medline]
Birren, S. J., Lo, L. and Anderson, D. J. (1993). Sympathetic neuroblasts undergo a developmental switch in trophic dependence. Development 119, 597-610.
Brambilla, R., Schnapp, A., Casagranda, F., Labrador, J. P., Bergemann, A. D., Flanagan, J. G., Pasquale, E. B. and Klein, R. (1995). Membrane-bound LERK2 ligand can signal through three different Eph- related receptor tyrosine kinases. EMBO J. 14, 3116-3126.[Abstract]
Breier, G., Breviario, F., Caveda, L., Berthier, R., Schnurch, H., Gotsch, U., Vestweber, D., Risau, W. and Dejana, E. (1996). Molecular cloning and expression of murine vascular endothelial-cadherin in early stage development of cardiovascular system. Blood 87, 630-641.
Brooks, P. C., Clark, R. A. F. and Cheresh, D. A. (1994). Requirement of vascular integrin vß3 for angiogenesis. Science 264, 569-571.[Medline]
Bruckner, K., Pasquale, E. B. and Klein, R. (1997). Tyrosine phosphorylation of transmembrane ligands for Eph receptors. Science 275, 1640-1643.
Brutsaert, D. L., Fransen, P., Andries, L. J., De Keulenaer, G. W. and Sys, S. U. (1998). Cardiac endothelium and myocardial function. Cardiovasc Res. 38, 281-290.[Medline]
Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, C. et al. (1996). Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435-439.[Medline]
Coffin, J. D. and Poole, T. J. (1988). Embryonic vascular development: immunohistochemical identification of the origin and subsequent morphogenesis of the major vessel primordia in quail embryos. Development 102, 735-748.[Abstract]
Daniel, T. O., Stein, E., Cerretti, D. P., St John, P. L., Robert, B. and Abrahamson, D. R. (1996). ELK and LERK-2 in developing kidney and microvascular endothelial assembly. Kidney Int. 50, S73-S81.
Davis, S., Aldrich, T. H., Jones, P. F., Acheson, A., Compton, D. L., Jain, V., Ryan, T. E., Bruno, J., Radziejewski, C., Maisonpierre, P. C. et al. (1996). Isolation of Angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87, 1161-1169.[Medline]
Dickson, M. C., Martin, J. S., Cousins, F. M., Kulkarni, A. B., Karlsson, S. and Akhurst, R. J. (1995). Defective haematopoiesis and vasculogenesis in transforming growth factor-beta 1 knock out mice. Development 121, 1845-1854.
Dodelet, V. C. and Pasquale, E. B. (2000). Eph receptors and ephrin ligands: embryogenesis to tumorigenesis. Oncogene 19, 5614-5619.[Medline]
Drake, C. J. and Fleming, P. A. (2000). Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood 95, 1671-1679.
Drescher, U., Kremoser, C., Handwerker, C., Loschinger, J., Noda, M. and Bonhoeffer, F. (1995). In vitro guidance of retinal ganglion cell axons by RAGS, a 25 kDa tectal protein related to ligands for Eph receptor tyrosine kinases. Cell 82, 359-370.[Medline]
Dumont, D. J., Yamaguchi, T. P., Conlon, R. A., Rossant, J. and Breitman, M. L. (1992). Tek, novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors. Oncogene 7, 1471-1480.[Medline]
Dumont, D. J., Gradwohl, G., Fong, G.-H., Puri, M. C., Gerstenstein, M., Auerbach, A. and Breitman, M. L. (1994). Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. Genes Dev. 8, 1897-1909.[Abstract]
Dumont, D. J., Fong, G.-H., Puri, M. C., Gradwohl, G., Alitalo, K. and Breitman, M. L. (1995). Vascularization of the mouse embryo:a study of flk-1, tek, tie, and vascular endothelial growth factor expression during development. Dev. Dyn. 203, 80-92.[Medline]
Durbin, L., Brennan, C., Shiomi, K., Cooke, J., Barrios, A., Shanmugalingam, S., Guthrie, B., Lindberg, R. and Holder, N. (1998). Eph signaling is required for segmentation and differentiation of the somites. Genes Dev. 12, 3096-3109.
Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., OShea, K. S., Powell-Braxton, L., Hillan, K. J. and Moore, M. W. (1996). Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380, 439-442.[Medline]
Fishman, G. I. (1997). Timing is everything in life conditional transgene expression in the cardiovascular system. Circ. Res. 82, 837-844.
Fishman, M. C. and Chien, K. R. (1997). Fashioning the vertebrate heart: earliest embryonic decisions. Development 124, 2099-2117.
Flamme, I., Frolich, T. and Risau, W. (1997). Molecular mechanisms of vasculogenesis and embryonic angiogenesis. J. Cell. Physiol. 173, 206-210.[Medline]
Folkman, J. and DAmore, P. A. (1996). Blood vessel formation: what is its molecular basis? Cell 87, 1153-1155.[Medline]
Friedlander, M., Brooks, P. C., Schaffer, R. W., Kincaid, C. M., Varner, J. A. and Cheresh, D. A. (1995). Definition of two angiogenic pathways by distinct v integrins. Science 270, 1500-1502.[Abstract]
Frisen, J., Yates, P. A., McLaughlin, T., Friedman, G. C., OLeary, D. D. M. and Barbacid, M. (1998). Ephrin-A5 (AL1/RAGS) is essentail for proper retinal axon guidance and topographic mapping in the mammalian visual system. Neuron 20, 235-243.[Medline]
Gale, N. W. and Yancopoulos, G. D. (1999). Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, Angiopoietins, and ephrins in vascular development. Genes Dev. 13, 1055-1066.
Gale, N. W., Baluk, P., Pan, L., Kwan, M., Holash, J., DeChiara, T. M., McDonald, D. M. and Yancopoulos, G. D. (2001). Ephrin-B2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth- muscle cells. Dev. Biol. 230, 151-160.[Medline]
Gerety, S. S., Wang, H. U., Chen, Z. F. and Anderson, D. J. (1999). Symetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development. Mol. Cell 4, 403-414.[Medline]
Gory-Faure, S., Prandini, M. H., Pointu, H., Roullot, V., Pignot-Paintrand, I., Vernet, M. and Huber, P. (1999). Role of vascular endothelial-cadherin in vascular morphogenesis. Development 126, 2093-2102.
Goumans, M. J., Zwijsen, A., van Rooijen, M. A., Huylebroeck, D., Roelen, B. A. and Mummery, C. L. (1999). Transforming growth factor-beta signalling in extraembryonic mesoderm is required for yolk sac vasculogenesis in mice. Development 126, 3473-3483.
Gupta, A. R., Dejneka, N. S., DAmato, R. J., Yang, Z., Syed, N., Maguire, A. M. and Bennett, J. (2001). Strain-dependent anterior segment neovascularization following intravitreal gene transfer of basic fibroblast growth factor (bFGF). J. Gene Med. 3, 252-259.[Medline]
Hanahan, D. (1997). Signaling vascular morphogenesis and maintenance. Science 277, 48-50.
Helbling, P. M., Saulnier, D. M. and Brandli, A. W. (2000). The receptor tyrosine kinase EphB4 and ephrin-B ligands restrict angiogenic growth of embryonic veins in Xenopus laevis. Development 127, 269-278.
Hellstrom, M., Kaln, M., Lindahl, P., Abramsson, A. and Betsholtz, C. (1999). Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vesselformation in the mouse. Development 126, 3047-3055.
Hirschi, K. K., Rohovsky, S. A., Beck, L. H., Smith, S. R. and DAmore, P. A. (1999). Endothelial cells modulate the proliferation of mural cell precursors via platelet-derived growth factor-BB and heterotypic cell contact. Circ. Res. 84, 298-305.
Holland, S. J., Gale, N. W., Mbamalu, G., Yancopoulos, G. D., Henkemeyer, M. and Pawson, T. (1996). Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands. Nature 383, 722-725.[Medline]
Kisanuki, Y. Y., Hammer, R. E., Miyazaki, J., Williams, S. C., Richardson, J. A. and Yanagisawa, M. (2001). Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230, 230-242.[Medline]
Kramer, R., Bucay, N., Kane, D. J., Martin, L. E., Tarpley, J. E. and Theill, L. E. (1996). Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development. Proc. Natl. Acad. Sci. USA 93, 4833-4838.
Krebs, L. T., Xue, Y., Norton, C. R., Shutter, J. R., Maguire, M., Sundberg, J. P., Gallahan, D., Closson, V., Kitajewski, J., Callahan, R. et al. (2000). Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 14, 1343-1352.
Krull, C. E., Lansford, R., Gale, N. W., Collazo, A., Marcelle, C., Yancopoulos, G. D., Fraser, S. E. and Bronner-Fraser, M. (1997). Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration. Curr. Biol. 7, 571-580.[Medline]
Lakso, M., Sauer, B., Mosinger, B., Jr., Lee, E. J., Manning, R. W., Yu, S. H., Mulder, K. L. and Westphal, H. (1992). Targeted oncogene activation by site-specific recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6232-6236.[Abstract]
Ma, Q., Chen, Z., del Barco Barrantes, I., de la Pompa, J. L. and Anderson, D. J. (1998). neurogenin1 is essential for the determination of neuronal precursors for proximal cranial sensory ganglia. Neuron 20, 469-482.[Medline]
Marchionni, M. A., Goodearl, A. D., Chen, M. S., Bermingham-McDonogh, O., Kirk, C., Hendricks, M., Danehy, F., Misumi, D., Sudhalter, J., Kobayashi, K. et al. (1993). Glial growth factors are alternatively spliced erbB2 ligands expressed in the nervous system. Nature 362, 312-318.[Medline]
Mellitzer, G., Xu, Q. and Wilkinson, D. G. (1999). Eph receptors and ephrins restrict cell intermingling and communication. Nature 400, 77-81.[Medline]
Meyer, D. and Birchmeier, C. (1995). Multiple essential functions of neuregulin in development. Nature 378, 386-390.[Medline]
Nagy, A. (2000). Cre recombinase: the universal reagent for genome tailoring. Genesis 26, 99-109.[Medline]
Ogawa, K., Pasqualini, R., Lindberg, R. A., Kain, R., Freeman, A. L. and Pasquale, E. B. (2000). The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization. Oncogene 19, 6043-6052.[Medline]
Oh, S. P., Seki, T., Goss, K. A., Imamura, T., Yi, Y., Donahoe, P. K., Li, L., Miyazono, K., ten Dijke, P., Kim, S. et al. (2000). Activin receptor-like kinase 1 modulates transforming growth factor- beta 1 signaling in the regulation of angiogenesis. Proc. Natl. Acad. Sci. USA 97, 2626-2631.
Orban, P. C., Chui, D. and Marth, J. D. (1992). Tissue- and site-specific DNA recombination in transgenic mice. Proc. Natl. Acad. Sci. USA 89, 6861-6865.[Abstract]
Oshima, M., Oshima, H. and Taketo, M. M. (1996). TGF-beta receptor type II deficiency results in defects of yolk sac hematopoiesis and vasculogenesis. Dev. Biol. 179, 297-302.[Medline]
Patan, S. (2000). Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. J. Neuro-Oncol. 50, 1-15.[Medline]
Pepper, M. S. (1997). Transforming growth factor-beta: vasculogenesis, angiogenesis, and vessel wall integrity. Cytokine Growth Factor Rev. 8, 21-43.[Medline]
Risau, W. and Flamme, I. (1995). Vasculogenesis. Annu. Rev. Cell Dev. Biol. 11, 73-91.[Medline]
Rohan, R. M., Fernandez, A., Udagawa, T., Yuan, J. and DAmato, R. J. (2000). Genetic heterogeneity of angiogenesis in mice. FASEB J. 14, 871-876.
Sakano, S., Serizawa, R., Inada, T., Iwama, A., Itoh, A., Kato, C., Shimizu, Y., Shinkai, F., Shimizu, R., Kondo, S. et al. (1996). Characterization of a ligand for receptor protein-tyrosine kinase HTK expressed in immature hematopoietic cells. Oncogene 13, 813-822.[Medline]
Sato, T. N., Tozawa, Y., Deutsch, U., Wolburg-Buchholz, K., Fujiwara, Y., Gendron-Maguire, M., Gridley, T., Wolburg, H., Risau, W. and Qin, Y. (1995). Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 376, 70-74.[Medline]
Schlaeger, T. M., Qin, Y., Fujiwara, Y., Magram, J. and Sato, T. N. (1995). Vascular endothelial cell lineage-specific promoter in transgenic mice. Development 121, 1089-1098.
Schlaeger, T. M., Bartunkova, S., Lawitts, J. A., Teichmann, G., Risau, W., Deutsch, U. and Sato, T. N. (1997). Uniform vascular-endothelial-cell-specific gene expression in both embryonic and adult transgenic mice. Proc. Natl. Acad. Sci. USA 94, 3058-3063.
Schor, A. M., Canfield, A. E., Sutton, A. B., Arciniegas, E. and Allen, T. D. (1995). Pericyte differentiation. Clin. Orthop. 313, 81-91.[Medline]
Shin, D., Garcia-Cardena, G., Hayashi, S., Gerety, S., Asahara, T., Stavrakis, G., Isner, J., Folkman, J., Gimbrone, M. A., Jr and Anderson, D. J. (2001). Expression of ephrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells, and marks subsets of microvessels at sites of adult neovascularization. Dev. Biol. 230, 139-150.[Medline]
Shutter, J. R., Scully, S., Fan, W., Richards, W. G., Kitajewski, J., Deblandre, G. A., Kintner, C. R. and Stark, K. L. (2000). Dll4, a novel Notch ligand expressed in arterial endothelium. Genes Dev. 14, 1313-1318.
Soriano, P. (1999). Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21, 70-71.[Medline]
Stein, E., Cerretti, D. P. and Daniel, T. O. (1996). Ligand activation of ELK receptor tyrosine kinase promotes its association with Grb10 and Grb2 in vascular endothelial cells. J. Biol. Chem. 271, 23588-23593.
Stein, E., Lane, A. A., Cerretti, D. P., Schoecklmann, H. O., Schroff, A. D., van Etten, R. L. and Daniel, T. O. (1998). Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses. Genes Dev. 12, 667-678.
Suri, C., Jones, P. F., Patan, S., Bartunkova, S., Maisonpierre, P. C., Davis, S., Sato, T. N. and Yancopoulos, G. D. (1996). Requisite role of Angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87, 1171-1180.[Medline]
Takai, N., Miyazaki, T., Fujisawa, K., Nasu, K. and Miyakawa, I. (2001). Expression of receptor tyrosine kinase EphB4 and its ligand ephrin-B2 is associated with malignant potential in endometrial cancer. Oncol. Rep. 8, 567-573.[Medline]
Tybulewicz, V. L., Crawford, C. E., Jackson, P. K., Bronson, R. T. and Mulligan, R. C. (1991). Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 65, 1153-1163.[Medline]
Wang, H. U. and Anderson, D. J. (1997). Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron 18, 383-396.[Medline]
Wang, H. U., Chen, Z. F. and Anderson, D. J. (1998). Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell 83, 741-753.
Wilkinson, D. G. (2000). Eph receptors and ephrins: regulators of guidance and assembly. Int. Rev. Cytol. 196, 177-244.[Medline]
Xu, Q., Alldus, G., Holder, N. and Wilkinson, D. G. (1995). Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain. Development 121, 4005-4016.
Xu, Q., Alldus, G., Macdonald, R., Wilkinson, D. G. and Holder, N. (1996). Function of the Eph-related kinase rtk1 in patterning of the zebrafish forebrain. Nature 381, 319-322.[Medline]
Xu, Q., Mellitzer, G., Robinson, V. and Wilkinson, D. G. (1999). In vivo cell sorting in complementary segmental domains mediated by Eph receptors and ephrins. Nature 399, 267-271.[Medline]
Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, S. J. and Holash, J. (2000). Vascular-specific growth factors and blood vessel formation. Nature 407, 242-248.[Medline]
Zinyk, D. L., Mercer, E. H., Harris, E., Anderson, D. J. and Joyner, A. L. (1998). Fate mapping of the mouse midbrain-hindbrain constriction using a site- specific recombination system. Curr. Biol. 8, 665-668.[Medline]