Application of infrared laser to the zebrafish vascular system:Gene induction, tracing, and ablation of single endothelial cells

Abstract

Objective-Infrared laser-evoked gene operator is a new microscopic method optimized to heat cells in living organisms without causing photochemical damage. By combining the promoter system for the heat shock response, infrared laser- evoked gene operator enables laser-mediated gene induction in targeted cells. We applied this method to the vascular system in zebrafish embryos and demonstrated its usability to investigate mechanisms of vascular morphogenesis in vivo. Approach and Results-We used double-transgenic zebrafish with fli1:nEGFP to identify the endothelial cells, and with hsp:mCherry to carry out single-cell labeling. Optimizing the irradiation conditions, we finally succeeded in inducing the expression of the mCherry gene in single targeted endothelial cells, at a maximum efficiency rate of 60%. In addition, we indicated that this system could be used for laser ablation under certain conditions. To evaluate infrared laser-evoked gene operator, we applied this system to the endothelial cells of the first intersegmental arteries, and captured images of the connection between the vascular systems of the brain and spinal cord. Conclusions-Our results suggest that the infrared laser-evoked gene operator system will contribute to the elucidation of the mechanisms underlying vascular morphogenesis by controlling spatiotemporal gene activation in single endothelial cells, by labeling or deleting individual vessels in living embryos. © 2013 American Heart Association, Inc.