Novel zebrafish model reveals a critical role for MAPK in lymphangiogenesis

Abstract

Purpose: Lymphatic disorders are poorly understood with few animal models. We designed a novel assay to measure lymphatic development using transgenic zebrafish with fluorescently labeled endothelial cells. Two major branches of the vascular endothelial growth factor receptor (VEGFR) signaling pathway were examined: the MAPK and PI3K pathways.

Methods: Direct visualization of lymphatic development was performed in control embryos or under chemical inhibition. Treatment involved a 6-hour pulse of inhibitor at 3 days postfertilization. Fish were analyzed for the presence of the thoracic duct (TD) at 4 days postfertilization (n > 100 specimens).

Results: Thoracic duct formation was prevented using selective inhibitors against kinases (MAPK, PI3K/TOR, or VEGFR). These kinases were important for TD formation because the lymphatic vessel failed to form in most of treated animals. Remarkably, MAPK pathway inhibition most robustly reduced lymphangiogenesis, demonstrated by a lack of lymphatic endothelial cells.

Conclusion: We conclude that MAPK pathway function downstream of the VEGFRs is crucial at the early stages of TD development. This study provides a novel animal model and a potential target pathway for further investigation. We suggest further examination of MAPK pathway deregulation as a potential mechanism underlying lymphatic disease in humans.

Figure 1. MAPK and PI3K Signaling Pathways

VEGFR2 and R3 can stimulate MAPK and PI3K signaling pathways in vitro. Inhibitors CI-1040, BEZ235 (BEZ), and PTK787/ZK2222584 (PTK) were employed to target the MAPK pathway, the PI3K pathway, or both pathways, respectively.

Figure 2. Formation of Thoracic Duct in Treated and Untreated Zebrafish Embryos

Embryos treated with MAPK (Mek1/2) pathway inhibitor CI-1040 developed progressive pericardial (black arrow) and diffuse (black arrowhead) edema over time (panels A-C). Microscopic magnification of boxed areas in A and B are noted. TD development was evaluated over a length of 6 ISVs and located between the dorsal aorta and posterior cardinal vein (labeled) (panel A′). Panel D shows a schematic indicating the thoracic duct in yellow. In fish treated with CI-1040, loss of TD formation and lack of presumed LECs (white arrows) were noted (panel B′). Compared with controls, all treatment groups exhibited statistical prevention of formation of TD (p<0.0001, Chi squared test). In addition, CI-1040 more strongly prevented formation compared with BEZ (p<0.0001, OR=3.9) Error bars indicate standard deviation between experiments (panel E). Scale bar A 300 μm; A′ 100 μm.