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Multiple factors are required to form functional lymphatic vessels. Here, we uncover an essential role for the secreted protein Svep1 and the transmembrane receptor Tie1 during the development of subpopulations of the zebrafish facial lymphatic network. This specific aspect of the facial network forms independently of Vegfc signalling, which otherwise is the most prominent signalling axis in all other lymphatic beds. Additionally, we find that multiple specific and newly uncovered phenotypic hallmarks of svep1 mutants are also present in tie1, but not in tie2 or vegfc mutants. These phenotypes are observed in the lymphatic vasculature of both head and trunk, as well as in the development of the dorsal longitudinal anastomotic vessel (DLAV) under reduced flow conditions. Furthermore, we show genetic interaction between svep1 and tie1 during the migration of parachordal lymphangioblasts (PLs). Therefore, our study demonstrates an important function for Tie1 signalling during lymphangiogenesis as well as blood vessel development in zebrafish and provides the first in vivo evidence for zebrafish Svep1 and Tie1 interaction. Since compound heterozygous mutations for SVEP1 and TIE2 have recently been reported in human glaucoma patients, our data have clinical relevance in demonstrating a role for Svep1 in Tie signalling in an in vivo setting.

The lymphatic vasculature is essential for tissue fluid homeostasis, immune cell surveillance and dietary lipid absorption, and has emerged as a key regulator of organ growth and repair1. Despite significant advances in our understanding of lymphatic function, the precise developmental origin of lymphatic endothelial cells (LECs) has remained a point of debate for over a century2-5. It is currently widely accepted that most LECs are derived from venous endothelium4,6, although other sources have been described, including mesenchymal cells3, hemogenic endothelium7 and musculoendothelial progenitors8,9. Here we show that the initial expansion of mammalian LECs is driven primarily by the in situ differentiation of specialized angioblasts and not migration from venous endothelium. Single-cell RNA sequencing and genetic lineage tracing experiments in mouse revealed a population of Etv2+Prox1+ lymphangioblasts that arise directly from paraxial mesoderm-derived progenitors. Conditional lineage labelling and morphological analyses showed that these specialized angioblasts emerge within a tight spatiotemporal window, and give rise to LECs in numerous tissues. Analysis of early LEC proliferation and migration supported these findings, suggesting that emergence of LECs from venous endothelium is limited. Collectively, our data reconcile discrepancies between previous studies and indicate that LECs form through both de novo specification from lymphangioblasts and transdifferentiation from venous endothelium.