Visualization of embryonic lymphangiogenesis advances the use of the zebrafish model for research in cancer and lymphatic pathologies

Abstract

Lymphangiogenesis induced during tumor growth contributes to metastasis. Genetic and chemical screens using the zebrafish model have the potential to enhance our understanding of lymphangiogenesis, and lead to the discovery of pharmacological agents with activity in the lymphatic system. Large-scale screening of lymphatic development in the whole zebrafish embryo requires a specific lymphatic endothelial cell marker. We isolated the zebrafish ortholog of Lyve1, and analyzed its expression pattern during embryogenesis, and under conditions where key regulators of lymphangiogenesis such as Prox1 and VegfC were depleted. Like humans, zebrafish embryos form lymph sacs, lymphangioblasts arise from venous endothelia, and they form asymmetric left and right collecting ducts. By monitoring the earliest lymphatic sprouting in the head, a pilot drug assay was performed showing rapamycin, an inhibitor of mammalian lymphangiogenesis, can also suppress zebrafish lymphangiogenesis. This work opens up a novel opportunity to further the understanding of, and potentially manipulate, human lymphangiogenesis. Developmental Dynamics 239:2128-2135,

Getting out and about: the emergence and morphogenesis of the vertebrate lymphatic vasculature

“…The recent generation of new, robust transgenic lines that label the venous and lymphatic vascular systems in zebrafish has uncovered previously uncharacterised lymphatic vascular beds and modes of embryonic lymphangiogenesis (Flores et al, 2010;Okuda et al, 2012). These lymphatic vessels include the facial lymphatics (FLs) [also observed previously (Yaniv et al, 2006;Bussmann et al, 2010)] (Fig.…”

An In Vivo Antilymphatic Screen in Zebrafish Identifies Novel Inhibitors of Mammalian Lymphangiogenesis and Lymphatic-Mediated Metastasis

“…LYVE-1 is extensively used as a marker for lymphatic vessels in mice and humans, and we found zebrafish lyve1 to be an excellent marker for lymphatic development as it is expressed in the veins and the developing lymphatic vessels (26). We have also shown that treatment of zebrafish embryos with known lymphatic inhibitors reduced or abolished the expression of lyve1 mRNA (25).…”

“…We recently identified and characterized the expression of the zebrafish ortholog of lymphatic vessel endothelial hyaluronan receptor 1 (lyve1; ref. 25). LYVE-1 is extensively used as a marker for lymphatic vessels in mice and humans, and we found zebrafish lyve1 to be an excellent marker for lymphatic development as it is expressed in the veins and the developing lymphatic vessels (26).…”

“…In situ hybridization was performed using a lyve1 antisense probe (25) in the Biolane HTI in situ robot (H€ olle and H€ uttner), as described previously (29).…”

“…Using our antilymphatic screening assay (see Supplementary Methods), we identified 12 compounds, a hit rate of 1.1%, which reduced or abolished lyve1 expression without adverse side-effects such as developmental delay or tissue necrosis. The mTOR inhibitor, rapamycin, was included as a positive control as it is a known inhibitor of both mammalian and zebrafish lymphangiogenesis (25,31,32). A secondary screen for thoracic duct formation using lyve1:egfp embryos (26) showed that six compounds displayed antilymphatic activity in zebrafish: the flavonoid kaempferol, the dihydroorotate dehydrogenase inhibitor leflunomide, 2 diphenylpiperazine calcium channel antagonists, flunarizine and cinnarizine and 2 statins, lovastastin, and simvastatin ( Fig.…”