Elucidating the mechanisms guiding lymphangiogenesis using the zebrafish model of lymphatic development

Abstract

Lymphatic vessels are important for tissue fluid homeostasis and immune cell trafficking. If lymphatic vessel growth or function is impaired, this results in the build-up of fluid (lymphoedema), while aberrant lymphatic growth can exacerbate many chronic inflammatory conditions and aid in the metastasis of solid tumours. Understanding the mechanisms that underpin lymphatic development could help develop treatments for these lymphatic-related diseases; however, the precise origin of lymphatic progenitors and the mechanisms that orchestrate lymphatic vessel formation are still unknown. Veins are widely recognised as the predominant source of lymphatic progenitors, however recent evidence suggests that they may have a 'dual origin'. Zebrafish are useful for studying the development of lymphatics because of the unique advantages they provide, including the ability to perform highresolution live imaging of developing vessels. The aim of this study was to use zebrafish to elucidate the cellular and molecular mechanisms guiding lymphatic development through the generation of photoconvertible fluorescent transgenics, time-lapse imaging, reverse genetics and immunofluorescence studies. Lineage tracing and live imaging confirmed that the zebrafish facial lymphatic network is initially formed from three distinct lymphatic progenitor populations through a 'relay-like' mechanism, whereby vessel migration and formation involves the sequential addition of lymphangioblasts (migratory lymphatic progenitors) to the growing vessel. In addition, this study is the first to describe a zebrafish lymphatic progenitor that is not derived from a vein, supporting the 'dual origin' theory of lymphatic development. Lymphatic specification was found to be heterogeneous across distinct progenitor populations, as each responds differently to molecular cues such as Vegfr3 signalling in relation to Prox1 induction, the master regulator of lymphatic fate. The response to molecular cues in the head is also distinct from the trunk, as Wnt/[beta]-catenin signalling was found to restrict lymphatic progenitor specification and proliferation in the head despite promoting it in the trunk. Finally, it was revealed that mdh1aa, a metabolic enzyme, may be involved in the regulation of lymphatic sprouting and migration in the zebrafish trunk. Overall, effective utilisation of the zebrafish model of lymphatic development has expanded our understanding of how lymphatic progenitors are specified, sprout and coalesce into lymphatic vessels.