MiniSOG2-mediated Specific Photoablation of Motor Neurons in Ascidian Embryos

Abstract

When understanding the neuronal function of a specific neural circuit, single-cell level photoablation of a targeted cell is one of the useful experimental approaches. This protocol describes a method to photoablate specific motor neurons via the mini singlet oxygen generator (miniSOG2), a light–oxygen–voltage (LOV)-based optogenetic tool used for ablating targeted cells in arbitrary areas. MiniSOG2 could induce the cell death pathway by generating reactive oxygen species (ROS) upon blue light illumination. Photoablation of a specific cell using the miniSOG2 was performed to show that, in Ciona intestinalis type A (Ciona robusta), a single pair of motor neurons, MN2/A10.64, is necessary to drive their tail muscle contraction. The membrane targeted miniSOG2 combined with neuron-specific promoter (pSP-Neurog::miniSOG2-CAAX) was electroplated into the Ciona egg and transiently expressed at specific neurons of the embryo. MN2 labeled with pSP-Neurog:mCherry-CAAX was irradiated using a 440-nm laser from the lateral side for 10 min to ablate its neural function. The behavior of the embryo before and after the irradiation was recorded with a high-speed camera.Graphical abstract: