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A genetic Xenopus laevis tadpole model to study lymphangiogenesis

Abstract

Lymph vessels control fluid homeostasis, immunity and metastasis. Unraveling the molecular basis of lymphangiogenesis has been hampered by the lack of a small animal model that can be genetically manipulated. Here, we show that Xenopus tadpoles develop lymph vessels from lymphangioblasts or, through transdifferentiation, from venous endothelial cells. Lymphangiography showed that these lymph vessels drain lymph, through the lymph heart, to the venous circulation. Morpholino-mediated knockdown of the lymphangiogenic factor Prox1 caused lymph vessel defects and lymphedema by impairing lymphatic commitment. Knockdown of vascular endothelial growth factor C (VEGF-C) also induced lymph vessel defects and lymphedema, but primarily by affecting migration of lymphatic endothelial cells. Knockdown of VEGF-C also resulted in aberrant blood vessel formation in tadpoles. This tadpole model offers opportunities for the discovery of new regulators of lymphangiogenesis.

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Figure 1: Expression of Prox1 and Msr in early tadpoles.
Figure 2: Development of the rostral lymph sac (RLS), lymph heart (LH) and caudal lymph vessels.
Figure 3: Lymphatic vascular development in tadpoles.
Figure 4: Impaired lymph vessel development in Prox1KD tadpoles.
Figure 5: Impaired lymph vessel development in VEGF-CKD tadpoles.

References

  1. Saharinen, P., Tammela, T., Karkkainen, M.J. & Alitalo, K. Lymphatic vasculature: development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol. 25, 387–395 (2004).

    CAS  Article  Google Scholar 

  2. Jain, R.K. Angiogenesis and lymphangiogenesis in tumors: insights from intravital microscopy. Cold Spring Harb. Symp. Quant. Biol. 67, 239–248 (2002).

    CAS  Article  Google Scholar 

  3. Alitalo, K. & Carmeliet, P. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell 1, 219–227 (2002).

    CAS  Article  Google Scholar 

  4. Asellius, G. Asellii Cremonensis antomici ticiensis qua sententiae anatomicae multae, nel perperam receptae illustrantur. De Lacteibus sive lacteis venis Quarto Vasorum Mesaroicum genere novo invente Gasp. (Mediolani, Milan, 1627).

    Google Scholar 

  5. Lawson, N.D. & Weinstein, B.M. Arteries and veins: making a difference with zebrafish. Nat. Rev. Genet. 3, 674–682 (2002).

    CAS  Article  Google Scholar 

  6. Daniels, C.B. et al. Regenerating lizard tails: a new model for investigating lymphangiogenesis. FASEB J. 17, 479–481 (2003).

    CAS  Article  Google Scholar 

  7. Wilting, J., Schneider, M., Papoutski, M., Alitalo, K. & Christ, B. An avian model for studies of embryonic lymphangiogenesis. Lymphology 33, 81–94 (2000).

    CAS  PubMed  Google Scholar 

  8. Olszewski, W. Regulation of water balance between blood and lymph in the frog, Rana pipiens. Lymphology 26, 154–155 (1993).

    CAS  PubMed  Google Scholar 

  9. Liu, Z.Y. & Casley-Smith, J.R. The fine structure of the amphibian lymph sac. Lymphology 22, 31–35 (1989).

    CAS  PubMed  Google Scholar 

  10. Hoyer, M. Untersuchungen ueber das Lymphgefaessystem der Froschlarven. Bull. Acad. Cracov. Teill II, 451–464 (1905).

    Google Scholar 

  11. Sabin, F.R. On the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig. Am. J. Anat. 1, 367–389 (1902).

    Article  Google Scholar 

  12. Wigle, J.T. & Oliver, G. Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769–778 (1999).

    CAS  Article  Google Scholar 

  13. Wigle, J.T. et al. An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype. EMBO J. 21, 1505–1513 (2002).

    CAS  Article  Google Scholar 

  14. Devic, E., Paquereau, L., Vernier, P., Knibiehler, B. & Audigier, Y. Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis . Mech. Dev. 59, 129–140 (1996).

    CAS  Article  Google Scholar 

  15. Cleaver, O., Tonissen, K.F., Saha, M.S. & Krieg, P.A. Neovascularization of the Xenopus embryo. Dev. Dyn. 210, 66–77 (1997).

    CAS  Article  Google Scholar 

  16. Levine, A.J., Munoz-Sanjuan, I., Bell, E., North, A.J. & Brivanlou, A.H. Fluorescent labeling of endothelial cells allows in vivo, continuous characterization of the vascular development of Xenopus laevis . Dev. Biol. 254, 50–67 (2003).

    CAS  Article  Google Scholar 

  17. Karkkainen, M.J. et al. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol. 5, 74–80 (2004).

    CAS  Article  Google Scholar 

  18. Baldwin, M.E. et al. Vascular endothelial growth factor D is dispensable for development of the lymphatic system. Mol. Cell. Biol. 25, 2441–2449 (2005).

    CAS  Article  Google Scholar 

  19. Achen, M.G. et al. Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc. Natl. Acad. Sci. USA 95, 548–553 (1998).

    CAS  Article  Google Scholar 

  20. Joukov, V. et al. A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J. 15, 290–298 (1996).

    CAS  Article  Google Scholar 

  21. Saaristo, A. et al. Adenoviral VEGF-C overexpression induces blood vessel enlargement, tortuosity, and leakiness but no sprouting angiogenesis in the skin or mucous membranes. FASEB J. 16, 1041–1049 (2002).

    CAS  Article  Google Scholar 

  22. Ober, E.A. et al. VEGF-C is required for vascular development and endoderm morphogenesis in zebrafish. EMBO Rep. 5, 78–84 (2004).

    CAS  Article  Google Scholar 

  23. Wilting, J. et al. Development of the avian lymphatic system. Microsc. Res. Tech. 55, 81–91 (2001).

    CAS  Article  Google Scholar 

  24. Newport, J. & Kirschner, M. A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell 30, 687–696 (1982).

    CAS  Article  Google Scholar 

  25. Nieuwkoop, P.J. & Faber, J. Normal table of Xenopus laevis (Daudin): A systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis (Garand Publishing, New York, 1994).

    Google Scholar 

  26. Harland, R.M. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36, 685–695 (1991).

    CAS  Article  Google Scholar 

  27. Carmeliet, P. et al. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat. Genet. 17, 439–444 (1997).

    CAS  Article  Google Scholar 

  28. Padera, T.P. et al. Lymphatic metastasis in the absence of functional intratumor lymphatics. Science 296, 1883–1886 (2002).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

A.N. is sponsored by a EU Sixth Framework Programme Marie Curie Intra European Fellowship; E.N. by the Fund for Scientific Research-Flanders (FWO); M.K. by the FCT (grant SFRH/BD/9349/2002, Portugal); M.S. and C.F. by the Deutsche Forschungsgemeinschaft (Germany). This work is supported, in part, by grant G.0567.05 from the FWO, by an unrestricted Bristol-Myers-Squibb grant, by a grant GOA2001/09 from Concerted Research Activities, Belgium and by grant CA-85140 from the US National Cancer Institute. The authors thank S. Tomarev and A. Ciau-Uitz for providing the rabbit anti-mouse Prox1 antibody and the Xenopus Fli and Msr cDNA, respectively, and K. Brepoels, A. Bouché, A. Claeys, M. De Mol, B. Hermans, S. Jansen, L. Kieckens, S. Louwette, A. Manderveld, W. Martens, L. Notebaert, A. Van Nuffelen, B. Vanwetswinkel (Leuven) and D. Fukumura (Boston) for their contribution.

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Correspondence to Peter Carmeliet.

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Supplementary information

Supplementary Fig. 1

Development of the rostral lymph sac and lymph heart. (PDF 560 kb)

Supplementary Fig. 2

Scheme of lymphatic cell migration (PDF 211 kb)

Supplementary Fig. 3

Identity and functionality of lymph vessels and heart. (PDF 277 kb)

Supplementary Fig. 4

Morpholinos block translation of Prox1 and VEGF-C. (PDF 290 kb)

Supplementary Fig. 5

Dose-dependence of Prox1 or VEGF-C knockdown. (PDF 83 kb)

Supplementary Fig. 6

Prox1 knockdown using the splice site morpholino. (PDF 401 kb)

Supplementary Movie 1

Intravital video-microscopy of VCLV and PCV. The first half of the movie shows the ventral caudal lymph vessel (VCLV), visualized by the progressive drainage of FITC-dextran after lymphangiography. In the second half of the movie, the microscope filters were switched to phase-contrast microscopy, revealing the posterior cardinal vein (PCV), containing flowing red blood cells. The movie was made using the same tadpole. Top of movie: dorsal side of the tadpole. (MOV 3125 kb)

Supplementary Movie 2

Intravital video-microscopy of lymph heart. Lymphangiography with FITC-dextran, showing the lymph heart, pumping the green fluorescent dye into the venous circulation through a set of valves. (AVI 2201 kb)

Supplementary Note (PDF 59 kb)

Supplementary Methods (PDF 92 kb)

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Ny, A., Koch, M., Schneider, M. et al. A genetic Xenopus laevis tadpole model to study lymphangiogenesis. Nat Med 11, 998–1004 (2005). https://doi.org/10.1038/nm1285

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