The role of vascular endothelial growth factor d in development of the facial lymphatic network in zebrafish

Abstract

The lymphatic vascular system is responsible for maintaining interstitial fluid homeostasis and it has important roles in lipid absorption and immune cell trafficking. The growth of lymphatic vessels, known as lymphangiogenesis, occurs in vertebrate embryonic development but it is also implicated in conditions of chronic inflammation and tumour metastasis. Vascular endothelial growth factor D (VEGFD) is a glycoprotein that signals migration, proliferation, and survival of lymphatic endothelial cells via activation of VEGF receptor 3, also known as FLT4. While tumour-derived VEGFD has been shown to induce lymphangiogenesis and subsequent tumour-cell metastasis, only the closely related protein VEGFC has been defined as essential for activation of VEGFR3 and subsequent lymphangiogenesis during development. Currently, based on mouse knockout data, it appears that Vegfd is not required for lymphatic vessel development. Our laboratory has recently generated an atlas of the embryonic lymphatic vasculature in zebrafish and described previously uncharacterised lateral, intestinal, and facial lymphatic (FL) networks. Surprisingly, antisense morpholino knockdown of vegfc, which is known to disrupt development of the trunk lymphatic network, did not impede the growth of the lateral facial lymphatic (LFL) vessel. In contrast, knockdown of flt4 inhibited initial development of the LFL; indicating this pathway is required in FL development and that Vegfd could potentially contribute to Flt4 activation. In support of this, we detected a spatiotemporal expression pattern of vegfd in the head that coincided with FL development. Knockdown of vegfd had no phenotype but when vegfd and vegfc were knocked down together, LFL development was impaired. These results show that vegfd can compensate in the absence of vegfc during formation of the LFL and provide the first indication of a role for vegfd in zebrafish lymphatic development. Overall, we have demonstrated a significant shift away from VEGFC as the primary ligand involved in lymphatic development and that we are progressing towards VEGFD not only being understood in a pathological context. Together, these conclusions will hold great relevance in how future therapeutics will target the VEGFR3 pathway in disease.