Exploring the guidance of facial lymphatic development in zebrafish

Abstract

Lymphatic vessels are essential for maintaining tissue fluid homeostasis, immune surveillance and lipid absorption. These vessels are largely derived from veins and develop to form a reproducible vascular network throughout the body. While the pathways that mediate lymphatic sprouting from veins are well established, there is little known about how lymphatic vessels are patterned during development. Many lymphatic vessels align with blood vessels and work in the zebrafish has shown that the development of trunk lymphatics require arterial guidance cues. However, a subset of zebrafish facial lymphatics such as the otolithic lymphatic vessel (OLV) and medial facial lymphatic (MFL) do not follow blood vessels, which suggests that non-vascular cues are involved in their guidance. This study revealed that the OLV is congruent with the middle lateral line nerve (nM), and time-lapse imaging showed its close association with the nM axon during development. Laser ablation of the middle lateral line ganglion (gM) and morpholino knockdown of neurog1 and foxi1 disrupted nM formation and concomitantly impaired OLV development, demonstrating that normal nM formation is required for OLV development. Further work revealed that the MFL conforms to the posterior surface of the hyosymplectic, interhyal and ceratohyal cartilages of the hyoid arch, and time-lapse imaging showed its preferential migration along these cartilages. Morpholino knockdown of tfap2a and kat6a disrupted hyoid cartilage formation and concomitantly impaired MFL development, suggesting that normal hyoid cartilage formation is required for MFL development. Vegfc is a growth factor required for lymphatic vessel sprouting and migration and this study revealed that the spatiotemporal expression of vegfc coincides with the posterior surface of the hyosymplectic cartilage, which implies that it may be the molecular cue required for cartilage guidance of the MFL. This work provides the first examples of sensory neurons and cartilage being involved in the guidance of lymphatic vessels. The findings offer an opportunity to investigate the molecular cues and processes mediating these interactions, to expand our current understanding of lymphatic development and lymphatic pathologies.