Utilising zebrafish as a model to investigate lymphangiogenesis and its contributions to disease

Abstract

Lymphatic vessels play an important role in the regulation of tissue fluid homeostasis, immune surveillance and dietary fat absorption. In addition, aberrant lymphatic development is associated with tumour metastasis and forms of chronic inflammation such as inflammatory bowel disease (IBD). Zebrafish are increasingly gaining importance in lymphatic research as they allow real-time visualisation of lymphatic vessel development (lymphangiogenesis). The aim of this work was to investigate the mechanisms of developmental and disease-associated lymphangiogenesis using novel zebrafish lymphatic reporter lines that utilised the promoter of lyve1, a gene previously shown to be expressed in zebrafish lymphatic vessels. Using these lymphatic reporters, a map describing lymphatic development in zebrafish up to 15 days post-fertilisation was established. This led to the discovery of three previously uncharacterised lymphatic networks in zebrafish: the facial lymphatics, the lateral lymphatics, and the intestinal lymphatics. Detailed characterisation of facial lymphatic development revealed that this lymphatic network develops using a novel mechanism, which involves vessel growth through a single vascular sprout followed by recruitment of lymphangioblasts that are derived from different veins. Using the lyve1 transgenic lines, a newly isolated oedematous mutant F186, was demonstrated to lack all lymphatic vessels. Subsequently, the causative mutation was shown to be a new mutant allele of ccbe1, a gene previously shown to be essential for lymphangiogenesis. The newly characterised intestinal lymphatic network was analysed to reveal that colitogenic challenge induces IBD-associated lymphangiogenesis in the zebrafish intestine. This shows for the first time that zebrafish can be used as a model for disease-associated inflammatory lymphangiogenesis. Zebrafish macrophages, neutrophils, and intestinal epithelial cells were shown to express vegfaa, vegfc, and vegfd. Finally, it was shown that Mycobacterium marinum-induced granulomas in larval zebrafish, an established model for tuberculosis, induce ectopic lymphatic vessels to form from the existing trunk lymphatic vasculature. This is the first description of lymphangiogenesis associated with tuberculous granulomas and suggests that the lymphatic vasculature may be involved in the progression of tuberculosis. In conclusion, the data and the tools generated in this study have expanded the zebrafish lymphatic field and have contributed in revealing novel aspects of developmental, and disease-associated lymphangiogenesis.