Tissue Regeneration Requires Edema Fluid Clearance by Compensatory Lymphangiogenesis in Zebrafish

Lymphangiogenesis is essential for tissue regeneration and edema clearance, and delayed or failed lymphangiogenesis is a critical cause of impaired healing. However, elucidating the dynamic changes in lymphangiogenesis during tissue regeneration in animal models in real-time has been challenging; thus, the mechanisms of compensatory lymphatic activation for edema clearance remain unclear. To address this, zebrafish were subjected to osmotic stress using a hypertonic solution (375 mOsm/L) and hypotonic solution (37.5 mOsm/L) to induce tissue damage followed by edema formation. Intravital imaging of Tg(mrc1a:egfp; kdrl:mcherry) larvae unveiled substantial lymphatic vessel remodeling during tissue regeneration. The increase of lymphatic endothelial progenitor cells accompanied by sustained expansion and remodeling of primary lymphatics during recovery suggested active lymphangiogenesis during tissue repair. We developed a novel method using translating ribosome affinity purification to scrutinize the translatome of lymphatic endothelial cells in vivo. This analysis revealed the upregulation of key pro-lymphangiogenic genes, notably vegfr2 and vegfr3, during tissue regeneration. Pharmacological inhibition of these Vegfr2/Vegfr3 prevented compensatory lymphangiogenesis and impaired healing. Our findings provide a new model of in vivo live imaging of regenerative lymphangiogenesis, presenting a novel approach to investigating lymphatic activation during tissue regeneration.