Lymphatic vessels are important for the regulation of tissue fluid homeostasis, immune surveillance and dietary fat absorption (Tammela and Alitalo, 2010). Aberrant lymphatic growth is associated with pathological conditions, including cancer metastasis and chronic inflammation, while a malfunctioning

INTRODUCTION Lymphatic vessels are important for the regulation of tissue fluid homeostasis, immune surveillance and dietary fat absorption (Tammela and Alitalo, 2010). Aberrant lymphatic growth is associated with pathological conditions, including cancer metastasis and chronic inflammation, while a malfunctioning lymphatic system results in lymphoedema (Alitalo, 2011). Fuelled by the identification of lymphatic specific markers and growth factors, extensive lymphatic research is being conducted with the hope of identifying therapeutic targets for these lymphatic abnormalities (Norrmen et al., 2011). Florence Sabin proposed the centrifugal growth of lymphatic vessels from the lymph sac, which originates from the venous vasculature system (Sabin, 1902). The venous origin of the lymph sacs has since been validated in mice and zebrafish (Kuchler et al., 2006; Yaniv et al., 2006; Srinivasan et al., 2007). Although subsequent lymphatic vessel formation is proposed to occur through both sprouting from the lymph sacs and from the direct delamination of lymphatic endothelial cells from the cardinal vein (Francois et al., 2012), little is known about how the lymph sacs remodel to form a complete lymphatic network. The optically transparent zebrafish embryo provides an ideal platform to investigate this. The formation of the thoracic duct, the major trunk lymphatic vessel in the zebrafish embryo, has been established as a model of lymphangiogenesis (Kuchler et al., 2006; Yaniv et al., 2006; Hogan et al., 2009b; Coffindaffer-Wilson et al., 2011). However, the remaining lymphatic network in the zebrafish embryo is poorly characterised and little is known about the development of lymphatic vessels outside the trunk. In this study, we generated transgenic lines using the zebrafish lymphatic vessel endothelial hyaluronan receptor 1 (lyve1) promoter. LYVE1 is one of the most specific and widely used mammalian lymphatic endothelial markers and its expression in a subpopulation of venous endothelial cells located in the cardinal vein provides the first indication of lymphatic endothelial commitment (Oliver, 2004; Tammela and Alitalo, 2010). Recently, we identified the zebrafish orthologue of LYVE1; zebrafish Lyve1 displays 34% amino acid similarity to human LYVE1 and is also expressed in the lymphatic vessels (Flores et al., 2010). These novel lyve1 transgenics enabled us to identify previously uncharacterised lymphatic vessels in the head, intestine and the superficial area of the trunk. The identification of these new lymphatic networks increases the versatility of the zebrafish as a tool with which to investigate lymphatic development. Using liveimaging approaches, we show that a facial lymphatic vessel, termed the lateral facial lymphatic (LFL), develops in a manner distinct to that previously described for the thoracic duct (TD). The LFL initially develops by the migration of a vascular sprout at the tip of the developing vessel; lymphangioblasts are recruited to this vascular tip to form a lymphatic vessel. Unlike the TD, we have shown that the lymphangioblasts that contribute to the LFL do not derive from a single source, showing that lymphatic vessel formation in zebrafish is more complex than previously thought.