Identifying structural and molecular cues required for facial lymphatic development in zebrafish

Abstract

The vertebrate lymphatic system plays an important role in the maintenance of body fluid homeostasis, immune surveillance and lipid transport. Lymphatic vessels develop through the synthesis of lymphatic vessels from the pre-existing venous blood vessels. Zebrafish is an ideal model for investigating lymphatic development due to the ease of genetic modification and the ability to visualise vascular growth in vivo using transparent larvae. One outstanding question in the field is how lymphatic vessels are patterned during development. It is known that nonlymphatic tissues such as motoneurons and blood vessels can direct lymphatic vessel development, but the molecular mechanisms behind lymphatic vessel guidance are not known. Recent work from our laboratory has identified the hyoid cartilage as a potential lymphatic guidance structure, as the medial facial lymphatic vessel (MFL) develops along this cartilage in the zebrafish head. Currently, it is unclear whether the hyoid cartilage is necessary for correct patterning of the MFL during development. Here, we confirmed the anatomical alignment between the hyoid cartilage and MFL via confocal microscopy. Hyoid cartilage development was disrupted by three different approaches: morpholino knockdown of two genes, kat6a and tfap2a, required for hyoid cartilage specification, gene mutation of kat6a and nitroreductasemediated cartilage ablation. All three experiments resulted in defective MFL elongation, demonstrating that the hyoid cartilage is required for the development of the MFL. This study also isolated pure cartilage cells via fluorescence-activated cell sorting (FACS) and compared the expression level of candidate lymphatic guidance genes between kat6a morphants and control morphants via qPCR. It was found that the mRNA abundance of vegfc, a vascular growth factor previously shown to be involved in lymphatic sprouting and migration, was significantly lower in kat6a morphants when compared to control morphants while ectopic overexpression of vegfc rescued the MFL inhibition in kat6a morphants. Taken together these data suggest that vegfc is the lymphatic guidance molecule expressed in hyoid cartilage. This study provides evidence for the requirement of hyoid cartilage during MFL development and suggests that vegfc is the molecule expressed by hyoid cartilage to direct MFL growth.