Activating MAP2K1 Mutation In Zebrafish Endothelial Cells Causes Arteriovenous Shunts
Christopher L. Sudduth, MD1, Nicola Blum, PhD1, Yu Sheng Cheng, BS1, Matthew P. Vivero, MD1, Patrick Smits, PhD1, Nathan D. Lawson, PhD2, Arin K. Greene, MD1.
1Boston Children's Hospital, Harvard Medical School, Boston, MA, USA, 2University of Massachusetts Medical School, Worcester, MA, USA.
PURPOSE: Arteriovenous malformation (AVM) is a sporadic vascular malformation defined by a nidus of irregular blood vessels connecting arteries to veins instead of a normal capillary bed. Somatic activating mutations in MAP2K1 cause extracranial AVM. The purpose of this study was to create an AVM animal model using zebrafish.
METHODS: Single-cell stage casper;Tg(gata1:dsred) zebrafish embryos were injected with 1 nL of transgenesis mixture: 100 ng/μL of either (1) Control (pTol2-Fli:GFP) or (2) Mutant (pTol2-Fli:GFP-kdrl:MAP2K1K57N) plasmid DNA + 40 ng/μL Tol2 transposase mRNA. Erythrocytes in these fish express the fluorescent protein dsRed. Embryos were anesthetized with 0.4 mg/mL Tricaine, embedded in 1% low-melt agarose and then imaged using a Zeiss LSM 800 Confocal Microscope or a Nikon SMZ18 Fluorescence Microscope. Confocal images were obtained using Zen Blue version 2.5. Embryos were imaged 72 hours post-fertilization. Blood flow was visualized using dsRed fluorescence.
RESULTS: Injection of MAP2K1K57N plasmid resulted in abnormal arteriovenous shunts (58/96 transgenic embryos, 60%). The phenotype consisted of either (1) a proximal shunt with blood flowing through a direct connection between proximal aortic vessels to the common cardinal vein and immediately back into the heart (39/58 embryos, 67%) or (2) a mid-trunk shunt between the dorsal artery and posterior cardinal vein (19/58 embryos, 33%). Shunts were not present in control zebrafish (n=65). Endothelial cells at the site of the shunt expressed high levels of the marker transgene confirming shunts contained mutant endothelial cells.
CONCLUSION: Zebrafish endothelial cells expressing mutant MAP2K1 form abnormal arteriovenous shunts. The phenotype recapitulates extracranial human AVM. Mutant MAP2K1 zebrafish are a promising animal model for testing pharmacotherapy to treat AVM.
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