Final Activity Report Summary - LYMPHANGIOGENOMICS (Development and use of a novel Xenopus model to study functional lymphangiogenomics)
An imbalance in lymphatic vessel growth contributes to the development of several serious and often incurable disorders, such as cancer, metastases, inflammatory diseases and lymphedema. In order to more effectively identify therapies for these conditions, it is critical to better define the molecular basis for how vessels grow, fail to grow or grow excessively. The traditional way to analyse gene function is by knocking the gene out in a mouse model, however this is very time-consuming and expensive. Therefore the use of small animal models such as tadpoles has recently become popular. These models allow studying one or many genes at the same time by genetic (morpholino knockdown technology) or pharmacological manipulation (drugs) in a semi high throughput way and on a more moderate budget. During the last years, zebrafish has proven to be an exceptional model to study blood vessel formation (angiogenesis). However, as zebrafish do not have an intricate lymphatic system this animal model is not useful to analyse lymphatic genes. Therefore there was an emerging need to develop a small animal model in which lymphangiogenesis (growth of lymphatic vessels) can be studied.
During this fellowship, the applicant has evaluated the suitability to use Xenopus tadpoles to study lymphangiogenesis. The development of the lymphatic system in growing tadpoles was characterised using in situ hybridisation. Results showed that by four days after fertilisation a lymphatic system with beating lymph hearts has formed. Dye, injected in the fin of the tadpoles, was absorbed by lymphatic vessels in the trunk, confirming their functionality. In addition, when known lymphatic genes were suppressed in the developing embryo by injections of antisense-morpholinos, embryos failed to develop functional lymphatic vessels and acquired lymphedema. These results are similar to those observed in gene deficient mice lacking lymphatic genes and humans with lymphatic malformations, and demonstrate that the Xenopus tadpole is a most suitable genetic animal model to study lymphangiogenesis (Ny et al., Nat Med. 2005;11(9):998-1004).
By performing morpholino injections in the tadpole, the applicant analysed the roles of several lymphangiogenic genes in and beyond lymphangiogenesis. In collaboration with Jean Léon Thomas, vascular endothelial growth factor-C (VEGF-C) was revealed to function as a growth factor selectively required by neural progenitor cells expressing its receptor VEGFR-3 (Le Bras B et al., Nat Neurosci. 2006;9(3):340-8). Furthermore, the lymphatic factor VEGF-D was found to be redundant for normal lymphatic development, which was in agreement with results observed in VEGF-D gene deficient mice generated in the lab. However, by suppressing two different genes simultaneous (via co-injections of morpholinos) the applicant observed that tadpoles injected with VEGF-D and VEGF-C or VEGF-D and transcription factor Prox1 morpholino developed edema and impaired vessel functions at morpholino concentrations that did not or only minimally affected lymphangiogenesis in the single knock-downs. The results indicate that VEGF-C, VEGF-D and Prox1 cooperatively regulate lymphangiogenesis (Koch et al., 2007 Manuscript in preparation).
By establishing protocols for treating tadpoles with chemical compounds the applicant enabled studies of the lymphatic receptor VEGFR-3 during lymphatic development (Ny A et al., 2007, manuscript in preparation). In addition in collaboration with Sanofi Aventis, the effect of a chemical multi-FGF receptor antagonist on lymphangiogenesis was studied (Bono et al., 2007, manuscript in preparation).
During this fellowship the applicant also established a transgenic Xenopus line expressing green fluorescent protein in blood and lymphatic vessels. Phenotypic analyses of lymphangiogenic candidate genes will greatly benefit from being carried out in this strain as live embryos can be observed at any time point by fluorescence microscopy.
During this fellowship, the applicant has evaluated the suitability to use Xenopus tadpoles to study lymphangiogenesis. The development of the lymphatic system in growing tadpoles was characterised using in situ hybridisation. Results showed that by four days after fertilisation a lymphatic system with beating lymph hearts has formed. Dye, injected in the fin of the tadpoles, was absorbed by lymphatic vessels in the trunk, confirming their functionality. In addition, when known lymphatic genes were suppressed in the developing embryo by injections of antisense-morpholinos, embryos failed to develop functional lymphatic vessels and acquired lymphedema. These results are similar to those observed in gene deficient mice lacking lymphatic genes and humans with lymphatic malformations, and demonstrate that the Xenopus tadpole is a most suitable genetic animal model to study lymphangiogenesis (Ny et al., Nat Med. 2005;11(9):998-1004).
By performing morpholino injections in the tadpole, the applicant analysed the roles of several lymphangiogenic genes in and beyond lymphangiogenesis. In collaboration with Jean Léon Thomas, vascular endothelial growth factor-C (VEGF-C) was revealed to function as a growth factor selectively required by neural progenitor cells expressing its receptor VEGFR-3 (Le Bras B et al., Nat Neurosci. 2006;9(3):340-8). Furthermore, the lymphatic factor VEGF-D was found to be redundant for normal lymphatic development, which was in agreement with results observed in VEGF-D gene deficient mice generated in the lab. However, by suppressing two different genes simultaneous (via co-injections of morpholinos) the applicant observed that tadpoles injected with VEGF-D and VEGF-C or VEGF-D and transcription factor Prox1 morpholino developed edema and impaired vessel functions at morpholino concentrations that did not or only minimally affected lymphangiogenesis in the single knock-downs. The results indicate that VEGF-C, VEGF-D and Prox1 cooperatively regulate lymphangiogenesis (Koch et al., 2007 Manuscript in preparation).
By establishing protocols for treating tadpoles with chemical compounds the applicant enabled studies of the lymphatic receptor VEGFR-3 during lymphatic development (Ny A et al., 2007, manuscript in preparation). In addition in collaboration with Sanofi Aventis, the effect of a chemical multi-FGF receptor antagonist on lymphangiogenesis was studied (Bono et al., 2007, manuscript in preparation).
During this fellowship the applicant also established a transgenic Xenopus line expressing green fluorescent protein in blood and lymphatic vessels. Phenotypic analyses of lymphangiogenic candidate genes will greatly benefit from being carried out in this strain as live embryos can be observed at any time point by fluorescence microscopy.