1 INSERM U36, Collège de France, 11, place Marcelin Berthelot, 75005 Paris, France
2 Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, 00014 Helsinki, Finland
*Author for correspondence (e-mail: anne.eichmann{at}college-de-france.fr)
Accepted 11 July 2002
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SUMMARY |
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Key words: Endothelial cell, Growth factor receptor, Lymphangiogenesis, Mouse
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INTRODUCTION |
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Several studies have shown that VEGF signals angiogenesis, while VEGFC and VEGFD induce lymphangiogenesis. Overexpression of VEGF in the skin of transgenic mice or on the chick chorioallantoic membrane stimulates growth of blood-vascular capillaries without affecting the lymphatic vessels (Detmar et al., 1998; Wilting et al., 1996
). Conversely, overexpression of VEGFC in the same mouse or chick models stimulates lymphangiogenesis with only a weak effect on blood-vascular EC (Jeltsch et al., 1997
; Oh et al., 1997
). Lymphangiogenesis could be selectively inhibited by overexpression of a soluble VEGFR3 (Makinen et al., 2001
). VEGFC and VEGFD have also been shown to stimulate metastasis via the lymphatic vessels (Karpanen et al., 2001
; Mandriota et al., 2001
; Skobe et al., 2001
; Stacker et al., 2001
). A role for VEGFR3 as the signaling receptor mediating VEGFC or VEGFD actions was suggested by overexpression of a VEGFC form that does not bind VEGFR2 (VEGF-C156S) in the skin of mice, which also stimulates lymphangiogenesis (Veikkola et al., 2001
). Moreover, mutations in the tyrosine kinase domain of VEGFR3 have been linked to human hereditary primary lymphoedema, and mice harboring an ENU-induced mutation in the kinase domain of VEGFR3 also develop lymphoedema (Karkkainen et al., 2000
; Karkkainen et al., 2001
).
Several members of the VEGF family have been recently shown to bind to the non-kinase neuropilin (NRP) receptors. This small family of type I transmembrane proteins includes NRP1 (NRP Mouse Genome Informatics) and NRP2. Neuropilins were initially discovered in the nervous system, where they function as receptors for the class III family of semaphorins (Sema) to mediate chemorepulsive guidance of developing axons (Chen et al., 1997; He and Tessier-Lavigne, 1997
; Kolodkin et al., 1997
). NRP1 and NRP2 show overlapping sema binding specificities in vitro: NRP1 binds with high affinity to Sema3a, Sema3c and Sema3f, while NRP2 is a high affinity receptor for Sema3c and Sema3f (Chen et al., 1997
; Feiner et al., 1997
). However, NRP1 function appears only necessary for Sema3a-mediated repulsive guidance events, both in vitro and in vivo, and genetic ablation of Nrp1 phenocopies the neuronal defects observed in Sema3a mutant mice (Kitsukawa et al., 1997
). Gene inactivation of Nrp2 results in a distinct neuronal phenotype compared with the Nrp1 mutation (Chen et al., 2000
; Giger et al., 2000
). Homozygous Nrp2 mutants specifically lose their response to repulsive guidance events mediated by Sema3f. Fasciculation and guidance of distinct subsets of cranial nerves are perturbed in Nrp1 and Nrp2 mutants: Nrp1/ showed deficiencies in the cranial nerves VII, IX and X, which were not affected in Nrp2/ mice. Conversely, cranial nerves III and IV, which were normal in Nrp1 mutants, showed abnormal projections in Nrp2/ animals (Chen et al., 2000
; Giger et al., 2000
; Kitsukawa et al., 1997
). Altogether, these results suggested specific, non-redundant functions for NRP1 and NRP2 in the nervous system.
Nrp1 mutant mice also showed severe defects in cardiovascular development, resulting in death of homozygous embryos by embryonic day (E) 14 (Kawasaki et al., 1999). Defects in vessel formation included failure of capillary ingrowth into the brain and abnormal formation of aortic arches and yolk-sac vasculature. These defects may be due to a requirement of NRP1 as a receptor for several members of the VEGF family, including the heparin-binding VEGF isoforms VEGF165 and VEGF145 as well as VEGFB, VEGFE and PlGF (for a review, see Neufeld et al., 2002
). NRP1 enhanced VEGF165 binding to VEGFR2 and VEGFR2-mediated chemotaxis response of EC (Miao et al., 1999
; Soker et al., 1998
). In the developing avian vascular system, Nrp1 showed specific expression in arterial EC (Moyon et al., 2001
; Herzog et al., 2001
).
NRP2 has also been shown to bind several VEGF family members, including VEGF165, VEGF145, PlGF and VEGF-C (Karkkainen et al., 2001) (reviewed by Neufeld et al., 2002
). A specific role for NRP2 in the vascular system has not been described previously. Indeed, many Nrp2 mutants are viable until adulthood and show a grossly normal cardiovascular system. We report that the formation of small lymphatic vessels and capillaries is abnormal in Nrp2 homozygous mice. In all tissues examined, including the heart, lung, diaphragm, gut and skin, these vessels are absent or severely reduced until postnatal stages. These observations show a selective requirement of NRP2 in lymphatic vessel development.
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MATERIALS AND METHODS |
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In situ hybridization
In situ hybridization has been described previously (Moyon et al., 2001,) except that 6 µg/ml proteinaseK was used. Probes were a 1.2 kb mouse Nrp2 fragment (Chen et al., 1997
), a 2.2 kb rat Nrp1 fragment (position 104-2388) (kindly provided by A. Chédotal) and a mouse Vegfr3 fragment (position 2411-4154).
Immunohistochemistry
Tissue biopsies were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Immunohistochemistry was performed using the Tyramide Signal Amplification system (TSATM, NENTM Life Science Products, Boston, MA). Peroxidase activity was developed with 3-amino-9-ethyl carbazole (Sigma). Antibodies were a monoclonal anti-VEGFR3 (Kubo et al., 2000), biotinylated polyclonal anti-mouse VEGFR3, anti-NRP2 and anti-VEGFC (R&D Systems, Oxon, UK), PECAM (Pharmingen, San Diego, CA) and FITC-conjugated anti-smooth muscle actin (Sigma). X-gal staining was carried out as described (Puri et al., 1995
).
Lymphatic vessel counts
We prepared serial 7.5 µm transverse sections between the neck and the tail of E17 embryos (n=5 for Nrp2/, n=4 for Nrp2+/, n=2 for Nrp2+/+), which were ordered such that each slide contained sections from embryos of each genotype at the same anteroposterior level. These slides were stained with anti-VEGFR3 antibody (see Fig. 5C,E,G) and examined under the microscope (x25 objective, final magnification x110). For body skin, the area counted spanned the dermis of the dorsal half of the embryo. In initial counts, different anteroposterior levels of the body axis were counted separately, i.e. between the forelimbs, around the waist and between the legs; no significant differences in these regions were observed and the data were pooled. For counts of hindlimb skin, the area considered was the dermis covering the entire hindlimb. We counted manually on every fourth section the number of VEGFR3-positive lymphatic profiles, a profile was counted as one regardless of its size (i.e. Fig. 5C Nrp2+/; 11 profiles counted). The length of the dermis was measured, its thickness was similar in all embryos analyzed (see Fig. 5C-H) and was therefore not taken into account. No significant differences in embryos of the same genotype were observed, and data for embryos of the same genotype were pooled. The count thus represents the mean±s.d. of the sum of all vessel profiles per cm of dorsal half of the dermis. For counts of subserosal VEFGFR3-positive lymphatic profiles, we proceeded in the same way.
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Studies on lymphatic transport
FITC-dextran (2000 kDa, 8 mg/ml, Sigma) was injected intradermally into the ear and staining of the lymphatic network was followed by fluorescence stereomicroscopy.
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RESULTS |
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Different tissues of Nrp2 mutants were examined on sections and by whole-mount X-gal staining. Serial transverse sections of E13 and E15 embryos (n=5 for Nrp2/, n=10 for Nrp2+/, n=3 for Nrp2+/+) were stained with PECAM antibodies, which revealed no differences in blood vessels and capillaries between Nrp2/ and Nrp2+/ or Nrp2+/+ animals (Fig. 4A-C). Staining with NRP2 and VEGFR-3 antibodies showed that jugular lymphatic vessels (Fig. 4D-I) as well as lymphatics of the thoracic duct were present and appeared normal. However, striking differences in the formation of lymphatic capillaries were noted between Nrp2/ and Nrp2+/ or Nrp2+/+ embryos from E13 until P3. In E13 Nrp2+/+ and Nrp2+/ embryos, numerous VEGFR3- and NRP2-positive capillaries were present in the dermis (Fig. 4D,E,G,H). These capillaries were absent or strongly reduced in number in the Nrp2/ littermates (Fig. 4F,I). NRP2 staining of sections of Nrp2/ embryos confirmed their genotype: intracellular punctuate staining was observed instead of membrane staining, reflecting the retention of mutant protein in the endoplasmic reticulum (Fig. 4I).
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The number of VEGFR3-positive lymphatic vessels present in the skin was counted on transverse serial sections of E17 embryos (n=5 for Nrp2/, n=4 for Nrp2+/, n=2 for Nrp2+/+) (see Materials and Methods). No significant difference in lymphatic vessel number was observed in Nrp2+/+ compared with Nrp2+/ skins (vessel number per cm sectioned body skin 20.6±1.1 in Nrp2+/+, 19.4±1.1 in Nrp2+/; hindlimb skin 31.7±5.3 in Nrp2+/+, 32.4±2.0 in Nrp2+/). In Nrp2/ embryos, the lymphatic vessel number was reduced three times in the body skin (vessel number per cm=6.1±1.7) and 1.8 times in the skin of the limb (18.0±2.3). To monitor the proliferation of lymphatic EC, we measured BrdU incorporation into the lymphatic vessels of the body skin of E17 embryos (n=5 for Nrp2/, n=3 for Nrp2+/, n=1 for Nrp2+/+). In the Nrp2+/+ and Nrp2+/ mice, 23.9±3.7% of VEGFR3-positive cells had incorporated BrdU (Fig. 6A,C), while only 13.6±3.4% of VEGFR3-positive cells were also BrdU positive in the Nrp2/ mutants (Fig. 6B,D). Thus, a reduction of about 1.8-fold was observed in the proliferation of skin lymphatic vessels in E17 Nrp2 mutants. By contrast, no obvious decrease in the number of BrdU-positive cells in the epidermis (Fig. 6A,B) or other tissues was detected in the Nrp2/ animals compared with Nrp2+/ or Nrp2+/+.
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Altogether, of 36 Nrp2/ hearts and diaphragms examined between E15 and P3, 32 hearts showed an absence of lymphatic capillaries, the remaining four resembled Nrp2+/ mice. All diaphragms showed absence of lymphatic vessels; so all the 36 Nrp2/ mice could be clearly distinguished from their Nrp2+/ littermates by X-gal staining of these two tissues. Hearts and diaphragms of Nrp2+/ mice (n=106) always showed a similar staining pattern (Fig. 7A,F). Staining with an antibody recognizing VEGF-C revealed labeling of the superficial epicardium in both Nrp2/ and Nrp2+/ mice (Fig. 7D,E), suggesting that the observed decrease in lymphatic vessel density was not due to decreased levels of this growth factor.
To analyze a possible phenotype in the lung, we performed X-gal staining, VEGFR3 whole-mount immunohistochemistry of lung slices (not shown) and staining of transverse lung sections (n=3 for Nrp2/, n=3 for Nrp2+/ sectioned at E15; n=1 for Nrp2/, n=1 for Nrp2+/, n=1 for Nrp2+/+ at E17; n=4 for Nrp2/, n=4 for Nrp2+/, n=2 for Nrp2+/+ at P0) with antibodies directed against NRP2, VEGFR-3 and PECAM. Lymphatics of the thoracic duct were present in the Nrp2/ mice at all stages examined (Fig. 8A,B). However, lymphatic capillaries, numerous in Nrp2+/ or Nrp2+/+ lungs, were strongly reduced in Nrp2/ lungs (Fig. 8A,B,E-G). This phenotype was observed from E15 onwards and correlated with the appearance of VEFGR3-positive capillaries in the lungs at E15. PECAM staining did not reveal any differences in the blood-vascular EC of the different animals (Fig. 8C,D). At birth, lymphatic vessels surrounding the main bronchi had formed in the Nrp2/ lungs and were stained with anti-NRP2 and VEGFR3 antibodies (Fig. 8E-G). However, VEGFR3-positive capillaries were still absent in the Nrp2/ mutants (Fig. 8E-G). In the subserosal plexus of the gut, a reduction in the number of lymphatic vessels was observed in E15 and E17 embryos (Fig. 8H,I). Counting of the number of VEGFR3-positive vessels in the subserosal plexus confirmed this reduction: 5.2±0.4 VEGFR3-positive vessels per cm sectioned guts were observed in Nrp2+/ and Nrp2+/+ animals (n=4 sectioned at E17), while 2.3±0.7 vessels were counted per cm sectioned guts in Nrp2/ littermates (n=5).
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DISCUSSION |
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The mechanisms responsible for the development of lymphatic vessels are currently not well understood. The first detectable lymphatic vessels in the embryo are the thoracic duct and jugular lymph sacs, which are thought to develop by sprouting from veins (Sabin et al., 1909). Indeed, labeling with lymphatic-specific markers such as Prox1 (Wigle and Oliver, 1999
) or VEGFR3 (Kaipainen et al., 1995
) are suggestive of a sprouting process. NRP2 is likely to be downstream of these initial events in the formation of lymphatic EC, as its expression becomes downregulated in veins after that of VEGFR3. Wigle et al. (Wigle et al., 2002
) have recently shown that in Prox1 mutant mice, the initial sprout formation from veins is not affected, but that sprout progression is impaired and that the lymphatic EC instead adopt a blood-vascular phenotype. How the remaining lymphatic vessels in the body develop is currently unknown. Quail-chick chimera experiments have suggested the participation of mesoderm-derived lymphangioblasts (Wilting et al., 2000
), but the extent of their contribution to the developing lymphatic vasculature is currently unknown. Our observations of NRP2 staining suggest that there is a phase of lymphatic EC development from pre-existing lymphatic EC, analogous to the classically defined angiogenesis process. NRP2 staining carried out at different stages of development showed a progressive coverage of, for example, the heart and the diaphragm with lymphatic EC. These vessels did not appear to develop by sprouting from veins; they may be formed by mesoderm-derived lymphangioblasts, but the most likely explanation is that they are formed by sprouting from pre-existing lymphatic vessels. In homozygous NRP2 mutants, this sprouting process appears affected, while the formation of the lymphatic thoracic duct and other collecting lymphatic vessels occurs normally. It may therefore be speculated that NRP2 function is not required for sprouting of lymphatic vessels from veins, but for the sprouting of lymphatic EC from pre-existing lymphatic EC. The Nrp2 mutants analyzed here were previously reported to maintain very low levels of wild-type transcripts (Chen et al., 2000
). Their neural phenotype was indistinguishable from a complete loss-of-function mutant generated by Giger et al. (Giger et al., 2000
). The vascular phenotype of this second Nrp2 mutant has not been reported yet.
In the developing avian vascular system, expression of Nrp1 and Nrp2 has recently been shown to localize to arteries and veins, respectively (Moyon et al., 2001; Herzog et al., 2001
). Preferential expression of NRP1 in developing retinal arteries has also been recently reported in mice (Stalmans et al., 2002
). The observations reported here confirm and extend these findings: at early developmental stages, Nrp1 and Nrp2 are expressed in respectively arterial and venous EC. At later developmental stages, NRP2 mRNA and protein expression in veins decreases, but remains high in lymphatic EC, which can be also identified by expression of VEGFR3. The different expression patterns suggest non-redundant functions of NRP1 and NRP2 in the vascular system. Interestingly, inactivation of both Nrp1 and Nrp2 genes leads to embryonic lethality at E8.5 (Takashima et al., 2002
). Embryos homozygous for one NRP mutation and heterozygous for the other NRP gene died at E10-E10.5 because of deficiencies in arterial and venous branching (Takashima et al., 2002
). Taken together with the specific expression of Nrp1 and Nrp2 in respectively arterial and venous EC, these results suggest a requirement of both NRP genes for correct arterial and venous patterning during early remodeling of the primary vascular plexus. At later developmental stages, the Nrp1 mutation mainly affects the arterial compartment (Kawasaki et al., 1999
), while we here report a specific requirement for NRP2 in lymphatic EC.
NRP2 is co-expressed with VEGFR-3 in lymphatic EC, although in mature vessels of adult skin its levels were decreased, and it has been shown to bind VEGFC (Karkkainen et al., 2001). It is therefore tempting to speculate that NRP2 may act as a co-receptor for VEGFR3 to mediate VEGFC-dependent lymphangiogenesis, analogous to NRP1, which enhances VEGF165 activity via VEGFR2. However, biochemical analysis of the signal transduction initiated by NRP2 has yet to be performed. Mutations in the tyrosine kinase domain of VEGFR3 lead to cutaneous aplasia of lymphatic vessels, causing the formation of edema in both mouse and humans (Karkkainen et al., 2000
; Karkkainen et al., 2001
). No signs of edema were apparent in NRP2 mutant mice, in spite of the reduction of lymphatic vessels in the skin and the internal organs. Larger collecting lymphatic vessels were, however, present in the mutant animals, which may be sufficient for tissue fluid drainage. Furthermore, from P7 onwards we observed growth of lymphatic vessels in the different organs of Nrp2/ animals. Regrowth of lymphatic vessels during postnatal development has also been observed in transgenic mice overexpressing a soluble VEGFR3 (Makinen et al., 2001
). The precise mechanisms regulating embryonic and adult physiological and pathological lymphangiogenesis remain to be fully explored.
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ACKNOWLEDGMENTS |
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REFERENCES |
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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.[CrossRef][Medline]
Chen, H., Chedotal, A., He, Z., Goodman, C. S. and Tessier-Lavigne, M. (1997). Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron 19, 547-559.[Medline]
Chen, H., Bagri, A., Zupicich, J. A., Zou, Y., Stoeckli, E., Pleasure, S. J., Lowenstein, D. H., Skarnes, W. C., Chedotal, A. and Tessier-Lavigne, M. (2000). Neuropilin-2 regulates the development of selective cranial and sensory nerves and hippocampal mossy fiber projections. Neuron 25, 43-56.[Medline]
Detmar, M., Brown, L. F., Schon, M. P., Elicker, B. M., Velasco, P., Richard, L., Fukumura, D., Monsky, W., Claffey, K. P. and Jain, R. K. (1998). Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice. J. Invest. Dermatol. 111, 1-6.[Abstract]
Erhard, H., Rietveld, F. J., Brocker, E. B., de Waal, R. M. and Ruiter, D. J. (1996). Phenotype of normal cutaneous microvasculature. Immunoelectron microscopic observations with emphasis on the differences between blood vessels and lymphatics. J. Invest. Dermatol. 106, 135-140.[Abstract]
Feiner, L., Koppel, A. M., Kobayashi, H. and Raper, J. A. (1997). Secreted chick semaphorins bind recombinant neuropilin with similar affinities but bind different subsets of neurons in situ. Neuron 19, 539-545.[Medline]
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.[CrossRef][Medline]
Giger, R. J., Cloutier, J. F., Sahay, A., Prinjha, R. K., Levengood, D., Moore, S. E., Pickering, S., Simmons, D., Rastan, S., Walsh, F. S. et al. (2000). Neuropilin-2 is required in vivo for selective axon guidance responses to secreted smeaphorins. Neuron 25, 29-41.[Medline]
He, Z. and Tessier-Lavigne, M. (1997). Neuropilin is a receptor for the axonal chemorepellent semaphorin III. Cell 90, 739-751.[Medline]
Herzog, Y., Kalcheim, C., Kahane, N., Reshef, R. and Neufeld, G. (2001). Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins. Mech. Dev. 109, 115-119.[CrossRef][Medline]
Jeltsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., Rauvala, H., Swartz, M., Fukumura, D., Jain, R. K. and Alitalo, K. (1997). Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 276, 1423-1425.
Kaipainen, A., Korhoonen, J., Mustonen, T., van Hinsbergh, V., Fong, G. H., Dumont, D., Breitman, M. L. and Alitalo, K. (1995). Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. USA 92, 3566-3570.[Abstract]
Karkkainen, M. J., Ferrell, R. E., Lawrence, E. C., Kimak, M. A., Levinson, K. L., McTigue, M. A., Alitalo, K. and Finegold, D. N. (2000). Missense mutations interfere with VEGFR-3 signaling in primary lymphoedema. Nat. Genet. 25, 153-159.[CrossRef][Medline]
Karkkainen, M. J., Saaristo, A., Jussila, L., Karila, K. A., Lawrence, E. C., Pajusola, K., Bueler, H., Eichmann, A., Kettunen, M. I., Ylä-Herttuala, S. et al. (2001). A model for gene therapy of human reditary lyphedema. Proc. Natl. Acad. Sci. USA 98, 12677-12682.
Karkkainen, M. J. and Alitalo, K. (2002). Lymphatic endothelial regulation, lymphoedema, and lymph node metastasis. Semin. Cell Dev. Biol. 13, 9-18.[CrossRef][Medline]
Karkkainen, M. J., Mäkinen, T. and Alitalo, K. (2002). Lymphatic endothelium: a new frontier of metastasis research. Nat. Cell Biol. 4, E2-E5.[CrossRef][Medline]
Karpanen, T., Egeblad, M., Karkkainen, M. J., Kubo, H., Yla-Herttuala, S., Jaattela, M. and Alitalo, K. (2001). Vascular endothelial growth factor C promotes tumor lymphangiogensis and intralymphatic tumor growth. Cancer Res. 61, 1786-1790.
Kawasaki, T., Kitsukawa, T., Bekku, Y., Matsuda, Y., Sanbo, M., Yagi, T. and Fujisawa, H. (1999). A requirement for neuropilin-1 in embryonic vessel formation. Development 126, 4895-4902.
Kitsukawa, T., Shimizu, M., Sanbo, M., Hirata, T., Taniguchi, M., Bekku, Y., Yagi, T. and Fujisawa, H. (1997). Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice. Neuron 19, 995-1005.[Medline]
Kolodkin, A. L., Levengood, D. V., Rowe, E. G., Tai, Y. T., Giger, R. J. and Ginty, D. D. (1997). Neuropilin is a semaphorin III receptor. Cell 90, 753-762.[Medline]
Kubo, H., Fujiwara, T., Jussila, L., Hashi, H., Ogawa, M., Shimizu, K., Awane, M., Sakai, Y., Takabayashi, A., Alitalo, K. et al. (2000). Involvement of vascular endothelial growth factor receptor-3 in maintenance of integrity of endothelial cell lining during tumor angiogenesis. Blood 96, 546-553.
Makinen, T., Jussila, L., Veikkola, T., Karpanen, T., Kettunen, T., Pulkkanen, K. J., Kauppinen, R., Jackson, D. G., Kubo, H., Nishikawa, S. I. et al. (2001). Inhibition of lymphangiogenesis with resulting lymphedema in transgenic mice expressing soluble VEGF receptor-3. Nat. Med. 7, 199-205.[CrossRef][Medline]
Mandriota, S. J., Jussila, L., Jeltsch, M., Compagni, A., Baetens, D., Prevo, R., Banerji, S., Huarte, J., Montesano, R., Jackson, D. G. et al. (2001). Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumor metastasis. EMBO J. 20, 672-682.
Miao, H. Q., Soker, S., Feiner, L., Alonso, J. L., Raper, J. A. and Klagsbrun, M. (1999). Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor. J. Cell Biol. 146, 233-242.
Moyon, D., Pardanaud, L., Yuan, L., Bréant, C. and Eichmann, A. (2001). Plasticity of endothelial cells during arterial-venous differentiation in the avian embryo. Development 128, 3359-3370.
Neufeld, G., Cohen, T., Gengrinovitch, S. and Poltorak, Z. (1999). Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 13, 9-22.
Neufeld, G., Cohen, T., Shraga, N., Lange, T., Kessler, O. and Herzog, Y. (2002). The neuropilins: multifunctional semaphoring and VEGF receptors that modulate axon guidance and angiogenesis. Trends Cardiovasc. Med. 12, 13-19.[CrossRef][Medline]
Oh, S. J., Jeltsch, M. M., Birkenhäger, R., McCarthey, J. E. G., Weich, H. A., Christ, B., Alitalo, K. and Wilting, J. (1997). VEGF and VEGF-C: specific induction of angiogenesis and lymphangiogenesis in the differentiated avian chorioallantoic membrane. Dev. Biol. 188, 96-109.[CrossRef][Medline]
Puri, M. C., Rossant, J., Alitalo, K., Bernstein, A. and Partanen, J. (1995). The receptor tyrosine kinase TIE is required for integrity and survival of vascular endothelial cells. EMBO J. 14, 5884-5891.[Abstract]
Sabin, F. R. (1909). The lymphatic system in human embryos, with a consideration of the system as a whole. Am. J. Anat. 9, 43-91.
Skobe, M., Hawighorst, T., Jackson, D. G., Prevo, R., Janes, L., Velasco, P., Riccardi, P., Alitalo, K., Claffey, K. and Detmar, M. (2001). Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis. Nat. Med. 7, 192-198.[CrossRef][Medline]
Soker, S., Takashima, S., Miao, H. Q., Neufeld, G. and Klagsbrun, M. (1998). Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell 92, 735-745.[Medline]
Stacker, S. A., Caesar, C., Baldwin, M. E., Thornton, G. E., Williams, R. A., Prevo, R., Jackson, D. G., Nishikawa, S., Kubo, H. and Achen, M. G. (2001). VEGF-D promotes the metastatic spread of tumor cells via the lymphatics. Nat. Med. 7, 186-191.[CrossRef][Medline]
Stalmans, I., Ng, Y. S., Rohan, R., Fruttiger, M., Bouche, A., Yuce, A., Fujisawa, H., Hermans, B., Shani, M., Jansen. S. et al. (2002). Arteriolar and venular patterning in retinas of mice selectively expressing VEGF isoforms. J. Clin. Invest. 109, 327-336.
Takashima, S., Kitakaze, M., Asakura, M., Asanuma, H., Sanada, S., Tashiro, F., Niwa, H., Miyazaki Ji, J., Hirota, S., Kitamura, Y. et al. (2002). Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis. Proc. Natl. Acad. Sci. USA 99, 3657-3662.
Veikkola, T., Karkkainen, M., Claesson-Welsh, L. and Alitalo, K. (2000). Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res. 60, 203-212.
Veikkola, T., Jussila, L., Makinen, T., Karpanen, T., Jeltsch, M., Petrova, T. V., Kubo, H., Thurston, G., McDonald, D. M., Achen, M. G. et al. (2001). Signalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice. EMBO J. 15, 1223-1231.[CrossRef]
Wigle, J. T. and Oliver, G. (1999). Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769-778.[Medline]
Wigle, J. T., Harvey, N., Detmar, M., Lagutina, I., Grosveld, G., Gunn, M. D., Jackson, D. G. and Oliver, G. (2002). An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 21, 1505-1513.
Wilting, J., Birkenhäger, R., Eichmann, A., Kurz, H., Martiny-Baron, G., Marmé, D., McCarthy, J., Christ, B. and Weich, H. (1996). VEGF 121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of the chorioallantoic membrane. Dev. Biol. 176, 76-85.[CrossRef][Medline]
Wilting, J., Eichmann, A. and Christ, B. (1997). The avian VEGF receptor homologues Quek1 and Quek2 in blood-vascular and lymphatic endothelial and non-endothelial cells during quail embryonic development. Cell. Tissue Res. 288, 207-223.[CrossRef][Medline]
Wilting, J., Papoutsi, M., Schneider, M. and Christ, B. (2000). The lymphatic endothelium of the avian wing is of somitic origin. Dev. Dyn. 217, 271-278.[CrossRef][Medline]