Development of the Zebrafish Lymphatic System Requires Vegfc Signaling

Abstract

Lymphangiogenesis results in the formation of a vascular network distinct from arteries and veins that serves to drain interstitial fluid from surrounding tissues and plays a pivotal role in the immune defense of vertebrates as well as in the progression of cancer and other diseases . In mammals, lymph vessels are lined by endothelial cells possibly sprouting from embryonic veins, and their development appears to be critically dependent on the function of PROX1 and VEGFC signaling . The existence of a lymphatic system in teleosts has been a matter of debate for decades. Here we show on the morphological, molecular, and functional levels that zebrafish embryos develop a lymphatic vasculature that serves to retrieve components of the interstitium to the lymph system. We demonstrate the existence of vessels that are molecularly and functionally distinct from blood vessels and show that the development of these vessels depends on Vegfc and VEGFR-3/Flt4 signaling. These findings imply that the molecular components controlling lymphangiogenesis in zebrafish and mammals are conserved and that the zebrafish lymphatic system develops early enough to allow in vivo observations, lineage tracing, and genetic as well as pharmacological screens.

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

“…The first compound, kaempferol, a natural flavonoid that can be isolated from many plant sources such as broccoli, apples, and tea (37), was able to inhibit secondary sprouting from the PCV in zebrafish embryos. This phenotype is identical to the phenotype observed when the Vegfr3/Vegfc signaling pathway is impaired (17,19), and in support of this observation, we find that kaempferol is able to inhibit human VEGFR2 and VEGFR3 at low micromolar concentrations. We also found that kaempferol had good efficacy at preventing mammalian lymphangiogenesis into Matrigel plugs and at reducing the density of tumor-associated lymphatics, as well as the frequency of lymph node metastases when dosed either as a prevention or intervention drug.…”

“…Lymphatic vessels can be readily observed in transparent embryos, and zebrafish lymphatic development shares the same molecular mechanisms as used by mammals (17)(18)(19). Zebrafish embryos, like cell-based assays, are also amenable to small-molecule drug screens (20).…”

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

“…More recent advances in understanding the molecular lymphangiogenic program have been achieved using small animal models including the zebrafish (Ny et al, 2005;Kuchler et al, 2006;Yaniv et al, 2006;Butler et al, 2009;Hogan et al, 2009). Lymphangiogenesis is primarily an embryonic process, and in adults only occurs in the settings of inflammation, tissue repair and during tumorigenesis (Cueni and Detmar, 2008;Mumprecht and Detmar, 2009).…”

Developmental and pathological lymphangiogenesis: from models to human disease

“…The importance of VEGF-C/VEGFR-3 pathway for lymphatic vascular development has been recently highlighted by studies in zebraWsh, which resolved a long standing controversy about the presence of lymphatic vessels in teleost Wshes (Küchler et al 2006;Vogel and Claviez 1981;Yaniv et al 2006). ZebraWsh lymphatic vessels express high levels of Prox1, Nrp2 (neuropilin-2), Vegfr3, and Angpt2 (angiopoietin-2) and, knock-down of Prox1 and Vegfc or inhibition of VEGFR-3 signaling by expression of VEGF-C trap VEGFR-3-Ig fusion protein prevents the formation of lymphatic vasculature (Küchler et al 2006;Yaniv et al 2006).…”

“…ZebraWsh lymphatic vessels express high levels of Prox1, Nrp2 (neuropilin-2), Vegfr3, and Angpt2 (angiopoietin-2) and, knock-down of Prox1 and Vegfc or inhibition of VEGFR-3 signaling by expression of VEGF-C trap VEGFR-3-Ig fusion protein prevents the formation of lymphatic vasculature (Küchler et al 2006;Yaniv et al 2006). Use of zebraWsh or Xenopus embryos as small vertebrate models oVers unparalled opportunities for identiWcation of novel regulators of lymphatic vascular development and detailed analysis using in vivo imaging approaches (Ny et al 2005;Yaniv et al 2006).…”