Loss of Endothelial Tie1 Receptor Impairs Lymphatic Vessel Development-Brief Report
Objective— Studies of Tie1 gene-targeted embryos have demonstrated loss of blood vessel integrity, but the relevance of Tie1 in lymphatic vasculature development is unknown. We tested the hypothesis that the swelling observed in Tie1 mutant embryos is associated with lymphatic vascular defects.
Methods and Results— We could extend the survival of the Tie1-deficient embryos in the ICR background, which allowed us to study their lymphatic vessel development. At embryonic day (E) 14.5, the Tie1−/− embryos had edema and hemorrhages and began to die. Immunohistochemical analysis revealed that they have abnormal lymph sacs. Tie1−/− mutants were swollen already at E12.5 without signs of hemorrhage. Their lymph sacs were abnormally patterned, suggesting that lymphatic malformations precede the blood vascular defects. We generated mice with a conditional Cre/loxP Tie1neo locus and found that the homozygous Tie1neo/neo hypomorphic embryos survived until E15.5 with lymphatic malformations resembling those seen in the Tie1−/− mutants.
Conclusion— Our data show that loss of Tie1 results in lymphatic vascular abnormalities that precede the blood vessel phenotype. These findings indicate that Tie1 is involved in lymphangiogenesis and suggest differential requirements for Tie1 signaling in the two vascular compartments.
During vertebrate embryonic development, blood vessels form by vasculogenesis and angiogenesis. These processes are regulated by vascular endothelial growth factors (VEGFs), angiopoietins (Ang), and other guidance cues.1–3 In lymphangiogenesis,4 a specialized subset of endothelial cells in the anterior cardinal vein migrate toward mesenchymal VEGF-C to form the primary lymph sacs by embryonic day (E) 12.05 and continue further to form the lymphatic capillaries.4,5 Insufficient lymphangiogenesis results in lymphedema, swelling of the limbs. Understanding the molecular basis of lymphatic dysfunction should enable the development of better therapies.
Targeted disruption of the Tie1 gene6 results in embryonic lethality by E13.5 due to severe edema, hemorrhage, and defective microvessel integrity.7,8 The importance of Tie1 in lymphatic vessels is unknown. Furthermore, no ligand has been found for Tie1.1 Here we report that Tie1 is involved in embryonic lymphatic vessel formation.
Materials and Methods
The mice used in this study are described in the supplemental materials (see http://atvb.ahajournals.org).
Whole-Mount Staining and Blood Vessel Quantification
Analysis of the blood vasculature is described in the supplemental materials.
X-Gal Staining and Immunohistochemistry
β-galactosidase activity in the vasculature of the Tie1lacZ/+ mice, immunohistochemistry, and microscopy techniques are described in the supplemental materials.
Analysis of Statistical Significance
Data sets were analyzed for significance using Student’s t test. P<0.05 was considered statistically significant.
We found that homozygous Tie1 targeted (Tie1−/−) embryos in a pure ICR genetic background survive longer (E 14.5) than in the reported mixed 129Sv/CD-1 background (E 13.5).8 At E13, Tie1−/− embryos in an ICR background were swollen and hemorrhagic when compared with Tie1+/− or Tie1+/+ littermates (Figure 1A and data not shown). At E12.5, the Tie1−/− embryos showed swelling in the dorsal and neck regions (Figure 1A, white arrows) without signs of hemorrhage, whereas at E11.5, no aberrant phenotype was noted (Figure 1A).
In the absence of an obvious blood vessel phenotype at E12.5 (Supplemental text 1 and Supplemental Figures I and II), we focused on the lymphatic vessels. Staining for β-galactosidase activity in Tie1+/− embryos8 confirmed that Tie1 is expressed in the lymph sacs (Supplemental text 2 and Supplemental Figure III). Staining of sections from E13.5 and E14.5 Tie1−/− embryos for the expression of lymphatic endothelial markers Prox1, LYVE-1, and VEGFR-3 (Figure 1B and Supplemental Figure IV and data not shown) showed abnormally patterned lymph sacs (Supplemental text 3). Importantly, this lymphatic phenotype was first evident in E12.5 Tie1−/− embryos when no bleeding was yet observed (Figure 1B).
We generated mice with a conditional Tie1 targeting locus (Cre/loxP Tie1neo; Supplemental text 4 and Figure 2A). In contrast to the Tie1neo/+ mice that were viable and fertile, the Tie1neo/neo embryos at E15.5 displayed prominent edema but no hemorrhages (Figure 2B and Supplemental text 5 and Supplemental Figure V). However, the lymph sacs were abnormally patterned, like in the Tie1−/− mutants (Figure 2C and Supplemental Figure VI). Importantly, Flp-mediated excision of Neo abolished the hypomorphic phenotype seen in the Tie1neo/neo embryos (Supplemental Figure VII).
Our results show that Tie1 gene targeting leads to lymphatic vascular malformations and edema in the embryos at a stage when no hemorrhages are observed.
All four known angiopoietin ligands bind to Tie2, the other member of the Tie receptor family.9 Genetic deletion of Tie2 or Ang1 and endothelial overexpression of Ang2 results in similar embryonic lethal phenotypes with severe defects in the remodeling of the primary blood vascular plexus.1 Ang1 is a Tie2 agonist that is crucial for blood vessel stabilization, whereas Ang2 is a context-dependent antagonist.1 Ang1 can induce Tie1 phosphorylation in endothelial cells and recruit both Tie1 and Tie2 to cell-cell contacts, whereas Ang2 seems to antagonize Ang1-induced Tie1 phosphorylation.10 Ang2-deficient mice show blood vascular and lymphatic patterning defects, whereas the insertion of Ang1 in the Ang2 locus restores the lymphatic but not the blood vascular defects11 (Supplemental text 6). Ang1 can also induce lymphangiogenesis in mice.12 These data suggest that both Ang1 and Ang2 act as Tie2 agonists in the lymphatic vessels.
Our findings indicate that the Tie1 receptor is dispensable for lymphatic endothelial cell commitment but is required for normal embryonic lymphangiogenesis. This, together with the differential actions of angiopoietins on lymphatic and blood vasculature, suggests that the Tie1 levels fine-tune the effects of the angiopoietins and that the early development of the lymphatic vasculature is affected even before obvious changes in the blood vasculature on the loss of Tie1 signals.
We thank Drs Urban Deutsch, Juha Partanen, Tatiana Petrova, and Tuomas Tammela for critical comments on the manuscript; Drs Kirsi Sainio, Heidi Stuhlmann, and Adriana Gittenberger-de Groot for helpful advice on the data; and Alun Parsons, Tapio Tainola, Katja Salo, and the Molecular Imaging Unit (University of Helsinki) for excellent technical assistance.
Sources of Funding
This work was supported by the Finnish Academy of Sciences, The Finnish Cancer Organizations, and The European Union (MicroEnviMet FP7/2007-2011 grant agreement No. 201279).
E.K. and M.W. were equal co-authors.
Received August 28, 2009; accepted October 30, 2009.
Larrivee B, Freitas C, Suchting S, Brunet I, Eichmann A. Guidance of vascular development: lessons from the nervous system. Circ Res. 2009; 104: 428–441.
Srinivasan RS, Dillard ME, Lagutin OV, Lin FJ, Tsai S, Tsai MJ, Samokhvalov IM, Oliver G. Lineage tracing demonstrates the venous origin of the mammalian lymphatic vasculature. Genes Dev. 2007; 21: 2422–2432.
Partanen J, Armstrong E, Makela TP, Korhonen J, Sandberg M, Renkonen R, Knuutila S, Huebner K, Alitalo K. A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol Cell Biol. 1992; 12: 1698–1707.
Lee HJ, Cho CH, Hwang SJ, Choi HH, Kim KT, Ahn SY, Kim JH, Oh JL, Lee GM, Koh GY. Biological characterization of angiopoietin-3 and angiopoietin-4. Faseb J. 2004; 18: 1200–1208.
Saharinen P, Eklund L, Miettinen J, Wirkkala R, Anisimov A, Winderlich M, Nottebaum A, Vestweber D, Deutsch U, Koh GY, Olsen BR, Alitalo K. Angiopoietins assemble distinct Tie2 signaling complexes in endothelial cell-cell and cell-matrix contacts. Nat Cell Biol. 2008; 10: 527–537.
Gale NW, Thurston G, Hackett SF, Renard R, Wang Q, McClain J, Martin C, Witte C, Witte MH, Jackson D, Suri C, Campochiaro PA, Wiegand SJ, Yancopoulos GD. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by angiopoietin-1. Dev Cell. 2002; 3: 411–423.
Tammela T, Saaristo A, Lohela M, Morisada T, Tornberg J, Norrmen C, Oike Y, Pajusola K, Thurston G, Suda T, Yla-Herttuala S, Alitalo K. Angiopoietin-1 promotes lymphatic sprouting and hyperplasia. Blood. 2005; 105: 4642–4648.