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A genetic Xenopus laevis tadpole model to study lymphangiogenesis

Nature Medicine volume 11, pages 998–1004 (2005)Cite this article

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.

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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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Author notes

  1. Annelii Ny, Marta Koch, Martin Schneider, Elke Neven and Ricky T Tong: These authors contributed equally to this work.

Authors and Affiliations

  1. Flanders Interuniversity Institute for Biotechnology, Center for Transgene Technology and Gene Therapy, Campus Gasthuisberg O&N, Herestraat 49, KULeuven, Leuven, B-3000

    Annelii Ny, Marta Koch, Martin Schneider, Elke Neven, Sunit Maity, Christian Fischer, Stephane Plaisance, Diether Lambrechts, Sven Terclavers, Malgorzata Ciesiolka, Wing Yan Man, Sabine Wyns, Kris Vleminckx, Désiré Collen, Mieke Dewerchin, Edward M Conway, Lieve Moons & Peter Carmeliet

  2. Department of Molecular Biomedical Research, University of Ghent, Technology Park 927, Ghent, B-9052, Belgium

    Annelii Ny, Marta Koch, Martin Schneider, Elke Neven, Sunit Maity, Christian Fischer, Stephane Plaisance, Diether Lambrechts, Sven Terclavers, Malgorzata Ciesiolka, Wing Yan Man, Sabine Wyns, Kris Vleminckx, Désiré Collen, Mieke Dewerchin, Edward M Conway, Lieve Moons & Peter Carmeliet

  3. Department of Radiation Oncology, Harvard Medical School, Edwin L. Steele Laboratory for Tumor Biology, Massachusetts General Hospital, 55 Fruit Street, Boston, 02114, Massachusetts, USA

    Ricky T Tong & Rakesh K Jain

  4. Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, Wolfgang-Pauli Strasse 10, HCI, Zurich, CH-8057, Switzerland

    Christophe Héligon, Roland Kälin, Irena Senn & André Brändli

  5. Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, 825 NE 13th Street, Oklahoma City, 73104, Oklahoma, USA

    Florea Lupu

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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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